Integrated sensor arrays for biological and chemical analysis

ABSTRACT

An apparatus comprising a chemical field effect transistor array in a circuit-supporting substrate is disclosed. The transistor array has disposed on its surface an array of sample-retaining regions capable of retaining a chemical or biological sample from a sample fluid. The transistor array has a pitch of 10 μm or less and a sample-retaining region is positioned on at least one chemical field effect transistor which is configured to generate at least one output signal related to a characteristic of a chemical or biological sample in such sample-retaining region.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/862,930 filed Sep. 23, 2015. U.S. application Ser. No. 14/862,930 isa continuation of U.S. application Ser. No. 14/599,948 filed Jan. 19,2015. U.S. application Ser. No. 14/599,948 is a continuation of U.S.application Ser. No. 13/125,133 filed Jul. 1, 2011, which issued as U.S.Pat. No. 8,936,763 on Jan. 20, 2015. U.S. Pat. No. 8,936,763 is a 371filing of International Application No. PCT/US2009/005745 filed Oct. 22,2009. PCT/US2009/005745 claims priority to U.S. application Ser. No.12/475,311 and to U.S. application Ser. No. 12/474,897; both filed onMay 29, 2009. U.S. Ser. No. 12/474,897 claims the benefit of U.S.Provisional Application Nos. 61/196,953 filed Oct. 22, 2008, 61/198,222filed Nov. 4, 2008, and 61/205,626 filed Jan. 22, 2009. All applicationslisted in this section are incorporated by reference herein, each intheir entirety.

FIELD OF THE DISCLOSURE

The present disclosure is directed generally to semiconductor chips formaking chemical measurements, and more particularly, to single chipISFET arrays (and arrays of single chip ISFET arrays) for monitoring oneor more analytes.

BACKGROUND

Rapid and accurate measurement of biological and chemical analytes isimportant in many fields, ranging from diagnostics, to industrialprocess control, to environmental monitoring, to scientific research.Chemically sensitive, and in particular, ion-sensitive field effecttransistors (“chemFETs” and “ISFETs” respectively) have been used forsuch measurements for many years, e.g. Bergveld, Sensors and Actuators,88: 1-20 (2003); Yuqing et al., Biotechnology Advances, 21: 527-534(2003); and the like. More recently, attempts have been made tofabricate arrays of such sensors using integrated circuit technologiesto obtain spatially distributed and multi-analyte measurements using asingle device, e.g., Yeow et al., Sensors and Actuators B 44: 434-440(1997); Martinoia et al., Biosensors & Bioelectronics, 16: 1043-1050(2001); Milgrew et al., Sensors and Actuators B 103: 37-42 (2004);Milgrew et al., Sensors and Actuators B, 111-112: 347-353 (2005); Hizawaet al., Sensors and Actuators B, 117: 509-515 (2006); Heer et al.,Biosensors and Bioelectronics, 22: 2546-2553 (2007); and the like. Suchefforts face several difficult technical challenges, particularly whenISFET sensor arrays have scales in excess of thousands of sensorelements and densities in excess of many hundreds of sensor elements permm² Such challenges include making large-scale arrays with sensorelements having uniform performance characteristics from sensor tosensor within the array, and making sensor elements with footprints onthe order of microns which are capable of generating signals detectableagainst a background of many noise sources from both the sensor arrayitself and a fluidics system that conveys reactants oranalyte-containing samples to the array, For ISFET arrays comprisingsensor elements with charge-sensitive components, such as floatinggates, the former challenge is exacerbated by the accumulation oftrapped charge in or adjacent to such components, which is a common sideproduct of semiconductor fabrication technologies. The latter challengeis exacerbated by the requirement that analytes of interest directly orindirectly generate a charged species that accumulates at or on acharge-sensitive component of the ISFET sensor. In very dense arrays,diffusion, reactivity of the analyte or its surrogate,cross-contamination from adjacent sensors, as well as electrical noisein the sample fluid can all adversely affect measurements. Theavailability of large-scale ISFET array that overcome these challengeswould be highly useful in the above fields, particularly where everhighly parallel multiplex chemical measurements are required, such as inthe large scale genetic analysis of genomes.

SUMMARY

Aspects of the invention relate in part to large arrays of chemFETs ormore specifically ISFETs for monitoring reactions, including for examplenucleic acid (e.g., DNA) sequencing reactions, based on monitoringanalytes present, generated or used during a reaction. More generally,arrays including large arrays of chemFETs may be employed to detect andmeasure static and/or dynamic amounts or concentrations of a variety ofanalytes (e.g., hydrogen ions, other ions, non-ionic molecules orcompounds, etc.) in a variety of chemical and/or biological processes(e.g., biological or chemical reactions, cell or tissue cultures ormonitoring, neural activity, nucleic acid sequencing, etc.) in whichvaluable information may be obtained based on such analyte measurements.Such arrays may be employed in methods that detect analytes and/ormethods that monitor biological or chemical processes via changes incharge at the surfaces of sensors in the arrays, either by directaccumulation of charged products or by indirect generation or capture ofcharged species related to the concentration or presence of an analyteof interest. The present invention is exemplified in a number ofimplementations and applications, some of which are summarized below.

In one aspect the invention is directed to an apparatus comprising achemical field effect transistor array in a circuit-supportingsubstrate, such transistor array having disposed on its surface an arrayof sample-retaining regions capable of retaining a chemical orbiological sample from a sample fluid, wherein such transistor array hasa pitch of 10 μm or less and each sample retaining region is positionedon at least one chemical field effect transistor which is configured togenerate at least one output signal related to a characteristic of achemical or biological sample in such sample-retaining region. In oneembodiment, the characteristic of said chemical or biological sample isa concentration or an amount of a charged species and wherein each ofsaid chemical field effect transistors is an ion-sensitive field effecttransistor having a floating gate with a dielectric layer on a surfacethereof, the dielectric layer contacting said sample fluid and beingcapable of accumulating charge in proportion to a concentration of thecharged species in said sample fluid. In one embodiment, such chargedspecies is a hydrogen ion such that the sensors measure changes in pH ofthe sample fluid in or adjacent to the sample-retaining region thereof.In an aspect of such embodiment, the dielectric layer has a thicknessselected to maximize capacitance thereacross, consistent with otherrequirements. Such thickness may be selected in the range of from 1 to1000 nanometers (nm), or in a range of from 10 to 500 nm, or in a rangeof from 20 to 100 nm.

In another aspect, the invention is directed to an integrated sensorarray that comprises a plurality of sensors formed in acircuit-supporting substrate, each sensor comprising a chemical fieldeffect transistor, the sensors being in a planar array of greater than256 sensors at a density greater than 100 sensors per mm², each sensorof the array being configured to provide at least one output signalrelated to a concentration or presence of a chemical or biologicalsample proximate thereto, such output signal being substantially thesame for each sensor of the array in response to the same concentrationor presence of the same chemical or biological sample. In one embodimentof this aspect, the integrated sensor array further includes a pluralityof sample-retaining regions in said circuit-supporting substrate, eachsample-retaining region on (or alternatively below or beside) andoperationally associated with at least one of said sensors. In anotherembodiment, such sample retaining regions are each microwells configuredto hold a sample within a volume of sample fluid. In another embodiment,sensors of the integrated sensor array detect or measure a concentrationof a charged species and each of the sensors is an ion-sensitive fieldeffect transistor having a floating gate with a dielectric layer on asurface thereof, the dielectric layer contacting a sample fluidcontaining said chemical or biological sample and being capable ofaccumulating charge in proportion to a concentration of the chargedspecies in the sample fluid adjacent thereto. The dielectric layer has athickness selected to maximize capacitance thereacross, consistent withother requirements.

In a further aspect, the invention is directed to a single chip chemicalassay device that comprises: (a) a sensor array formed in a circuitsupporting substrate, each sensor of the array comprising a chemicalfield-effect transistor and being configured to provide at least oneoutput signal related to a concentration or presence of a chemical orbiological sample proximate thereto, such output signal beingsubstantially the same for each sensor of the array in response to thesame concentration or presence of the same chemical or biologicalsample; (b) a plurality of sample retaining regions in the circuitsupporting substrate, each sample-retaining region disposed on at leastone sensors; and (c) control circuitry in the circuit supportingsubstrate coupled to the sensor array to receive samples of the outputsignals from said chemical field effect transistors at a rate of atleast one frame per second.

In still another aspect the invention is directed to a single chipnucleic acid assay device that comprises: (a) a sensor array formed in acircuit supporting substrate, each sensor of the array comprising achemical field-effect transistor and being configured to provide atleast one output signal related to a concentration or presence of achemical or biological sample proximate thereto, such output signalbeing substantially the same for each sensor of the array in response tothe same concentration or presence of the same chemical or biologicalsample; (b) a plurality of sample retaining regions in the circuitsupporting substrate, each sample-retaining region disposed on at leastone sensors; (c) supports, including solid supports such as particulatesolid supports, disposed on the sample-retaining regions, each supporthaving a concatemerized template attached thereto; and (d) controlcircuitry in the circuit supporting substrate coupled to the sensorarray to receive samples of the output signals from said chemical fieldeffect transistors at a rate of at least one frame per second. Supportsmay include beads, including beads having solid or porous cores and/orsolid or porous surfaces, microspheres, microparticles, gelmicrodroplets and other separable particulate supports for attaching DNAtemplates, particularly as clonal populations. Such supports may be onthe order of microns or nanometers, depending on the application. Forexample, the beads may be microbeads or they may be nanobeads.

These above-characterized aspects, as well as other aspects, of thepresent invention are exemplified in a number of illustratedimplementations and applications, some of which are shown in the figuresand characterized in the claims section that follows. However, the abovesummary is not intended to describe each illustrated embodiment or everyimplementation of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead being placed upon generallyillustrating the various concepts discussed herein.

FIG. 1 generally illustrates a nucleic acid processing system comprisinga large scale chemFET array, according to one inventive embodiment ofthe present disclosure.

FIG. 2 illustrates one column of an chemFET array similar to that shownin FIG. 1, according to one inventive embodiment of the presentdisclosure.

FIG. 3 shows a composite cross-sectional view of multiple neighboringpixels illustrating a layer-by-layer view of pixel fabrication accordingto another inventive embodiment of the present disclosure.

FIGS. 4A-4L are top views of patterns corresponding layers of materiallaid down in the fabrication of sensors shown in FIG. 3.

FIG. 5A-5B illustrates process steps for fabricating an array with thindielectric layers on sensor floating gates, and a dielectric layercomprising a charge-sensitive layer and an adhesion layer.

FIG. 6 is a block diagram of an embodiment of the electronic componentsof one embodiment of the invention.

FIG. 7 is an exemplary timing diagram for components shown in FIG. 6.

FIG. 8A is a high-level, partially block, partially circuit diagramshowing a basic passive sensor pixel in which the voltage changes on theISFET source and drain inject noise into the analyte, causing errors inthe sensed values.

FIG. 8B is a high-level partially block, partially circuit diagramshowing a basic passive sensor pixel in which the voltage changes on theISFET drain are eliminated by tying it to ground, the pixel output isobtained via a column buffer, and CDS is employed on the output of thecolumn buffer to reduce correlated noise.

FIG. 8C is a high-level partially block, partially circuit diagramshowing a two transistor passive sensor pixel in which the voltagechanges on the ISFET drain and source are substantially eliminated, thepixel output is obtained via a buffer, and CDS is employed on the outputof the column buffer to reduce correlated noise.

FIG. 9 is a cross-sectional view of a flow cell.

FIGS. 10A and B are graphs showing a trace from an ISFET device (A) anda nucleotide readout (B) from a sequencing reaction of a 23-mersynthetic oligonucleotide.

FIGS. 11A and B are graphs showing a trace from an ISFET device (A) anda nucleotide readout (B) from a sequencing reaction of a 25-mer PCRproduct.

DETAILED DESCRIPTION

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention. For example, the microelectronicsportion of the apparatus and array is implemented in CMOS technology forpurposes of illustration. It should be appreciated, however, that thedisclosure is not intended to be limiting in this respect, as othersemiconductor-based technologies may be utilized to implement variousaspects of the microelectronics portion of the systems discussed herein.Guidance for making arrays of the invention is found in many availablereferences and treatises on integrated circuit design and manufacturingand micromachining, including, but not limited to, Allen et al., CMOSAnalog Circuit Design (Oxford University Press, 2″d Edition, 2002);Levinson, Principles of Lithography, Second Edition (SPIE Press, 2005);Doering and Nishi, Editors, Handbook of Semiconductor ManufacturingTechnology, Second Edition (CRC Press, 2007); Baker, CMOS CircuitDesign, Layout, and Simulation (IEEE Press, Wiley-Interscience, 2008);Veendrick, Deep-Submicron CMOS ICs (Kluwer-Deventer, 1998); Cao,Nanostructures & Nanomaterials (Imperial College Press, 2004); and thelike, which relevant parts are hereby incorporated by reference.

In one aspect the invention is directed to integrated sensor arrays(e.g., a two-dimensional array) of chemically-sensitive field effecttransistors (chemFETs), where the individual chemFET sensor elements or“pixels” of the array are configured to detect analyte presence (orabsence), analyte levels (or amounts), and/or analyte concentration in asample fluid. “Sample fluid” means a fluid which is used to deliversample or reagents to a sample-retaining region or to remove products orreactants from sample-retaining regions, such as a wash fluid of amulti-step reaction. Sample fluids may remain the same or may change inthe course of an analytical process, or processes, taking place on anarray. Analytical processes detected or monitored by arrays of theinvention include chemical and/or biological processes (e.g., chemicalreactions, cell cultures, neural activity, nucleic acid sequencingreactions, etc.) occurring in proximity to the array. Examples ofchemFETs contemplated by various embodiments discussed in greater detailbelow include, but are not limited to, ion-sensitive field effecttransistors (ISFETs) and enzyme-sensitive field effect transistors(EnFETs). In one exemplary implementation, one or more microfluidicstructures is/are fabricated above the chemFET sensor array to providefor retention, containment and/or confinement of a biological orchemical reaction in which an analyte of interest may be captured,produced, or consumed, as the case may be. Such structures defineregions on an array referred to herein as “sample-retaining regions.”For example, in one implementation, the microfluidic structure(s) may beconfigured as one or more wells (or microwells, or reaction chambers, orreaction wells as the terms are used interchangeably herein) disposedabove one or more sensors of the array, such that the one or moresensors over which a given well is disposed detect and measure analytepresence, level, and/or concentration in the given well. Preferably,there is a 1:1 ratio of wells and sensors.

A chemFET array according to various inventive embodiments of thepresent disclosure may be configured for sensitivity to any one or moreof a variety of analytes. In one embodiment, one or more chemFETs of anarray may be particularly configured for sensitivity to one or moreanalytes, and in other embodiments different chemFETs of a given arraymay be configured for sensitivity to different analytes. For example, inone embodiment, one or more sensors (pixels) of the array may include afirst type of chemFET configured to be sensitive to a first analyte, andone or more other sensors of the array may include a second type ofchemFET configured to be sensitive to a second analyte different fromthe first analyte. In one embodiment, the first and second analytes maybe related to each other. As an example, the first and second analytesmay be byproducts of the same biological or chemical reaction/processand therefore they may be detected concurrently to confirm theoccurrence of a reaction (or lack thereof). Such redundancy ispreferable in some analyte detection methods. Of course, it should beappreciated that more than two different types of chemFETs may beemployed in any given array to detect and/or measure different types ofanalytes, and optionally to monitor biological or chemical processessuch as binding events. In general, it should be appreciated in any ofthe embodiments of sensor arrays discussed herein that a given sensorarray may be “homogeneous” and thereby consist of chemFETs ofsubstantially similar or identical type that detect and/or measure thesame analyte (e.g., pH or other ion concentration), or a sensor arraymay be “heterogeneous” and include chemFETs of different types to detectand/or measure different analytes. In another embodiment, the sensors inan array may be configured to detect and/or measure a single type (orclass) of analyte even though the species of that type (or class)detected and/or measured may be different between sensors. As anexample, all the sensors in an array may be configured to detect and/ormeasure nucleic acids, but each sensor detects and/or measures adifferent nucleic acid.

As used herein, an array is a planar arrangement of elements such assensors or wells. The array may be one or two dimensional. A onedimensional array is an array having one column (or row) of elements inthe first dimension and a plurality of columns (or rows) in the seconddimension. An example of a one dimensional array is a 1×5 array. A twodimensional array is an array having a plurality of columns (or rows) inboth the first and the second dimensions. The number of columns (orrows) in the first and second dimensions may or may not be the same. Anexample of a two dimensional array is a 5×10 array.

Accordingly, one embodiment is directed to an apparatus, comprising anarray of CMOS-fabricated sensors, each sensor comprising onechemically-sensitive field effect transistor (chemFET) and occupying anarea on a surface of the array of 10 μm² or less, 9 μm² or less, 8 μm²or less, 7 μm² or less, 6 μm² or less, 5 μm² or less, 4 μm² or less 3μm² or less, or 2 μm² or less. Another embodiment of the invention isdirected to an apparatus comprising a planar array of CMOS-fabricatedsensors in a density of at least 100 sensors/mm², or at least 250sensors/mm², or at least 1000 sensors/mm², or at least 5000 sensors/mm²,or at least 10000 sensors/mm².

Another embodiment is directed to an apparatus, comprising an array ofCMOS fabricated sensors, each sensor comprising one chemically-sensitivefield effect transistor (chemFET). The array of CMOS-fabricated sensorsincludes more than 256 sensors, and a collection of chemFET outputsignals from all chemFETs of the array constitutes a frame of data. Theapparatus further comprises control circuitry coupled to the array andconfigured to generate at least one array output signal to providemultiple frames of data from the array at a frame rate of at least 1frame per second. In one aspect, the frame rate may be at least 10frames per second. In another aspect, the frame rate may be at least 20frames per second. In yet other aspects, the frame rate may be at least30, 40, 50, 70 or up to 100 frames per second. Chemical or biologicalphenomena measured by the sensors may result in electrical signals,usually current or voltage level changes, having a wide variety ofdurations and amplitudes. In some embodiments, such signals may have aduration in the range of a few milliseconds, e.g. 10 msec, to manyseconds, e.g. 10-20 sec. For cyclical or sequential processes monitoredby arrays, such signals may be substantially repeated over an intervalof minute, hours, or days. Moreover, the signal may have superimposedvarious types of noise, including flicker noise from the array itself,and thermal noise, particularly from the sample fluid. The controlcircuitry of the apparatus is configured to sample each signal from eachsensor in the array for a complete frame of data. Such sampling may befor a short duration, e.g. 1-100 μsec, or the like, so that completeframes of data may be readout in the frame per second values listedabove.

As explained more fully below, arrays of the invention have beenfabricated in a manner that reduces the effects of trapped charge, whichincludes lack of stability and uniformity of sensor-to-sensor responsesto the same or similar sensing conditions by sensor in the same array.In one embodiment, such responses may be measured by exposing sensors ofan array to predefined changes in pH. In one embodiment, responses of atleast 95 percent of the sensors in an array are substantially linearover a range of from 7 to 9 pH and has a voltage output signal with asensitivity of at least 40 m V/pH unit. In another embodiment, at least98 percent of sensor of an array have such performance. “Substantiallylinear” means that measured pH values over such pH range has acoefficient of variation of deviation from linearity of at least 10percent, or at least 5 percent, or at least 2 percent. In anotherembodiment, at least 95 percent of sensors of such arrays each have aresponse time to a change of pH of less than 200 msec.

Another embodiment is directed to a sensor array, comprising a pluralityof electronic sensors arranged in a plurality of rows and a plurality ofcolumns. Each sensor comprises one chemically-sensitive field effecttransistor (chemFET) configured to provide at least one and in someinstances at least two output signals representing a presence and/or aconcentration of an analyte proximate to a surface of the array. Foreach column of the plurality of columns, the array further comprisescolumn circuitry configured to provide a constant drain current and aconstant drain-to-source voltage to respective chemFETs in the column,the column circuitry including two operational amplifiers and adiode-connected FET arranged in a Kelvin bridge configuration with therespective chemFETs to provide the constant drain-to-source voltage.

Another embodiment is directed to a sensor array, comprising a pluralityof electronic sensors arranged in a plurality of rows and a plurality ofcolumns. Each sensor comprises one chemically-sensitive field effecttransistor (chemFET) configured to provide at least one output signaland in some instances at least two output signals representing aconcentration of ions in a solution proximate to a surface of the array.The array further comprises at least one row select shift register toenable respective rows of the plurality of rows, and at least one columnselect shift register to acquire chemFET output signals from respectivecolumns of the plurality of columns.

The apparatus and devices of the invention can be used to detect and/ormonitor interactions between various entities. These interactionsinclude biological and chemical reactions and may involve enzymaticreactions and/or non-enzymatic interactions such as but not limited tobinding events. As an example, the invention contemplates monitoringenzymatic reactions in which substrates and/or reagents are consumedand/or reaction intermediates, byproducts and/or products are generated.An example of a reaction that can be monitored according to theinvention is a nucleic acid synthesis method such as one that providesinformation regarding nucleic acid sequence. This reaction will bediscussed in greater detail herein.

Apparatus

Apparatus of the invention can vary widely depending on the analytebeing detected, whether assay reactions with auxiliary reagents arerequired, and whether sequential or cyclical reactions are required. Inone aspect, apparatus of the invention comprises a sensor array and anarray of sample-retaining regions on a surface thereof for retainingbiological or chemical analytes delivered to the surface by a samplefluid. In one embodiment, sample-retaining regions are integral with thesensor array and may have a wide variety of formats. Such regions may bedefined by chemically reactive group on the surface of the sensor array,by binding compounds attached to the surface of the sensor array whichare specific for predetermined analytes, by regions of hydrophobicity orhydrophilicity, or by spatial features such as microwells, cavities,weirs, dams, reservoirs, or the like. In additional embodiments,apparatus of the invention may comprise sample carriers, such as beads,particles, gel microdroplets, or other supports, structures orsubstances which hold analytes of interest and which may be delivered tosample-retaining regions by a sample fluid. Such sample carriers mayinclude binding moieties or reactive groups to permit attachment tosample-retaining regions. Such attachment may be specific wherein suchbinding moieties or reactive groups form linkages with onlycomplementary binding compounds or functionalities, or the attachmentmay be random where a sample carrier has a substantially equallikelihood of being retained in any sample-retaining region of an array.As described more fully below, in one embodiment, sample-retainingregions are arrays of microwells that each have walls and an interiorfor physically retaining sample, analyte and/or one or more samplecarriers.

As is exemplified below, sample fluid and samples or analytes may bedelivered to retaining regions of a sensor array in several ways. Wheresensor arrays are employed as one-use sample characterization devices,such as with process, environmental or cellular monitoring, sample maybe delivered by emersion, pipetting, pouring, or by other directmethods. Where sensor arrays are employed in sequential or cyclicalreactions, such as in DNA sequencing, sample fluidic, including nucleicacid templates, reagents, wash solutions, and the like, may be deliveredby a fluidic system under computer control. For such latterapplications, embodiments of the invention may further include a flowcell integrated with the sample-retaining regions and sensor array. Asdescribed more fully below, in one embodiment, such flow cell deliverssample fluids (including assay reactants, buffers, and the like) tosample-retaining regions under controlled conditions, which may includelaminar flow, constant flow rate at each sample-retaining region,controlled temperature, minimization of bubbles or other flowdisruptions, and the like. In one aspect, a flow cell of an apparatus ofthe invention comprises an inlet, an outlet, and an interior space,which when the flow cell is in communication with, for example sealinglybonded to, the arrays of sample retaining regions and sensors forms achamber that is closed except for the inlet and outlet. In someembodiments, the device is manufactured such that the flow cell and oneor both the arrays are integral to each other. In other embodiments, theflow cell is sealingly bonded to the arrays. Either embodiment willprevent fluid leakage, which, among other possible hazards, wouldintroduce electrical noise into the sample fluid. In one aspect, theapparatus of the invention includes a reference electrode in fluidcontact with the sample fluid so that during operation an electricalpotential difference is established between the reference electrode andthe sensors of the array.

An exemplary apparatus is shown in FIG. 1 which is adapted for nucleicacid sequencing. In the discussion that follows, the chemFET sensors ofthe array are described for purposes of illustration as ISFETsconfigured for sensitivity to static and/or dynamic ion concentration,including but not limited to hydrogen ion concentration. However, itshould be appreciated that the present disclosure is not limited in thisrespect, and that in any of the embodiments discussed herein in whichISFETs are employed as an illustrative example, other types of chemFETsmay be similarly employed in alternative embodiments, as discussed infurther detail below. Similarly it should be appreciated that variousaspects and embodiments of the invention may employ ISFETs as sensorsyet detect one or more ionic species that are not hydrogen ions.

The system 1000 includes a semiconductor/microfluidics hybrid structure300 comprising an ISFET sensor array 100 and a microfluidics flow cell200. In one aspect, the flow cell 200 may comprise a number of wells(not shown in FIG. 1) disposed above corresponding sensors of the ISFETarray 100. In another aspect, the flow cell 200 is configured tofacilitate the sequencing of one or more identical template nucleicacids disposed in the flow cell via the controlled and orderedintroduction to the flow cell of a number of sequencing reagents 272(e.g., dATP, dCTP, dGTP, dTTP (generically referred to herein as dNTP),divalent cations such as but not limited to Mg²⁺, wash solutions, andthe like).

As illustrated in FIG. 1, the introduction of the sequencing reagents tothe flow cell 200 may be accomplished via one or more valves 270 and oneor more pumps 274 that are controlled by a computer 260. A number oftechniques may be used to admit (i.e., introduce) the various processingmaterials (i.e., solutions, samples, reaction reagents, wash solutions,and the like) into the wells of such a flow cell. As illustrated in FIG.1, reagents including dNTP may be admitted to the flow cell (e.g., viathe computer controlled valve 270 and pumps 274) from which they diffuseinto the wells, or reagents may be added to the flow cell by other meanssuch as an ink jet. In yet another example, the flow cell 200 may notcontain any wells, and diffusion properties of the reagents may beexploited to limit cross-talk between respective sensors of the ISFETarray 100, or nucleic acids may be immobilized on the surfaces ofsensors of the ISFET array 100.

The flow cell 200 in the system of FIG. 1 may be configured in a varietyof manners to provide one or more analytes (or one or more reactionsolutions) in proximity to the ISFET array 100. For example, a templatenucleic acid may be directly attached or applied in suitable proximityto one or more pixels of the sensor array 100, or in or on a supportmaterial (e.g., one or more “beads”) located above the sensor array butwithin the reaction chambers, or on the sensor surface itself.Processing reagents (e.g., enzymes such as polymerases) can also beplaced on the sensors directly, or on one or more solid supports (e.g.,they may be bound to the capture beads or to other beads) in proximityto the sensors, or they may be in solution and free-flowing. It is to beunderstood that the device may be used without wells or beads.

In the system 1000 of FIG. 1, according to one embodiment the ISFETsensor array 100 monitors ionic species, and in particular, changes inthe levels/amounts and/or concentration of ionic species, includinghydrogen ions. In important embodiments, the species are those thatresult from a nucleic acid synthesis or sequencing reaction.

Via an array controller 250 (also under operation of the computer 260),the ISFET array may be controlled so as to acquire data (e.g., outputsignals of respective ISFETs of the array) relating to analyte detectionand/or measurements, and collected data may be processed by the computer260 to yield meaningful information associated with the processing(including sequencing) of the template nucleic acid.

With respect to the ISFET array 100 of the system 1000 shown in FIG. 1,in one embodiment the array 100 is implemented as an integrated circuitdesigned and fabricated using standard CMOS processes (e.g., 0.35micrometer process, 0.18 micrometer process), comprising all the sensorsand electronics needed to monitor/measure one or more analytes and/orreactions. With reference again to FIG. 1, one or more referenceelectrodes 76 to be employed in connection with the ISFET array 100 maybe placed in the flow cell 200 (e.g., disposed in “unused” wells of theflow cell) or otherwise exposed to a reference (e.g., one or more of thesequencing reagents 172) to establish a baseline against which changesin analyte concentration proximate to respective ISFETs of the array 100are compared. The reference electrode(s) 76 may be electrically coupledto the array 100, the array controller 250 or directly to the computer260 to facilitate analyte measurements based on voltage signals obtainedfrom the array 100; in some implementations, the reference electrode(s)may be coupled to an electric ground or other predetermined potential,or the reference electrode voltage may be measured with respect toground, to establish an electric reference for ISFET output signalmeasurements, as discussed further below.

More generally, a chemFET array according to various embodiments of thepresent disclosure may be configured for sensitivity to any one or moreof a variety of analytes. In one embodiment, one or more chemFETs of anarray may be particularly configured for sensitivity to one or moreanalytes and/or one or more binding events, and in other embodimentsdifferent chemFETs of a given array may be configured for sensitivity todifferent analytes. For example, in one embodiment, one or more sensors(pixels) of the array may include a first type of chemFET configured tobe sensitive to a first analyte, and one or more other sensors of thearray may include a second type of chemFET configured to be sensitive toa second analyte different from the first analyte. In one exemplaryimplementation, both a first and a second analyte may indicate aparticular reaction such as for example nucleotide incorporation in asequencing-by-synthesis method. Of course, it should be appreciated thatmore than two different types of chemFETs may be employed in any givenarray to detect and/or measure different types of analytes and/or otherreactions. In general, it should be appreciated in any of theembodiments of sensor arrays discussed herein that a given sensor arraymay be “homogeneous” and include chemFETs of substantially similar oridentical types to detect and/or measure a same type of analyte (e.g.,hydrogen ions), or a sensor array may be “heterogeneous” and includechemFETs of different types to detect and/or measure different analytes.

In other aspects of the system shown in FIG. 1, one or more arraycontrollers 250 may be employed to operate the ISFET array 100 (e.g.,selecting/enabling respective pixels of the array to obtain outputsignals representing analyte measurements). In various implementations,one or more components constituting one or more array controllers may beimplemented together with pixel elements of the arrays themselves, onthe same integrated circuit (IC) chip as the array but in a differentportion of the IC chip, or off-chip. In connection with array control,analog-to-digital conversion of ISFET output signals may be performed bycircuitry implemented on the same integrated circuit chip as the ISFETarray, but located outside of the sensor array region (locating theanalog to digital conversion circuitry outside of the sensor arrayregion allows for smaller pitch and hence a larger number of sensors, aswell as reduced noise). In various exemplary implementations discussedfurther below, analog-to-digital conversion can be 4-bit, 8-bit, 12-bit,16-bit or other bit resolutions depending on the signal dynamic rangerequired.

In general, data may be removed from the array in serial or parallel orsome combination thereof. On-chip controllers (or sense amplifiers) cancontrol the entire chip or some portion of the chip. Thus, the chipcontrollers or signal amplifiers may be replicated as necessaryaccording to the demands of the application. The array may, but need notbe, uniform. For instance, if signal processing or some other constraintrequires instead of one large array multiple smaller arrays, each withits own sense amplifiers or controller logic, that is quite feasible.

Having provided a general overview of the role of a chemFET (e.g.,ISFET) array 100 in an exemplary system 1000 for measuring one or moreanalytes, following below are more detailed descriptions of exemplarychemFET arrays according to various inventive embodiments of the presentdisclosure that may be employed in a variety of applications. Again, forpurposes of illustration, chemFET arrays according to the presentdisclosure are discussed below using the particular example of an ISFETarray, but other types of chemFETs may be employed in alternativeembodiments. Also, again, for purposes of illustration, chemFET arraysare discussed in the context of nucleic acid sequencing applications,however, the invention is not so limited and rather contemplates avariety of applications for the chemFET arrays described herein.

Sensor Layout and Array Fabrication

Methods of sensor layout design and array fabrication are described inRothberg et al., U.S. patent publications 2009/0026082 and 2009/0127589.In particular, techniques are disclosed for reducing or eliminatingtrapped charged during; accordingly, these references are incorporatedby reference. In one aspect, the sensor design and signal readoutcircuitry may be employed in the present invention. For example, in oneembodiment shown in FIG. 2, each chemFET of an array comprises afloating gate structure, and a source and a drain having a firstsemiconductor type and fabricated in a region having a secondsemiconductor type, wherein there is no electrical conductor thatelectrically connects the region having the second semiconductor type toeither the source or the drain. Each sensor consists of three fieldeffect transistors (FETs) including the chemFET, and each sensorincludes a plurality of electrical conductors electrically connected tothe three FETs. The three FETs are arranged such that the plurality ofelectrical conductors includes no more than four conductors traversingan area occupied by each sensor and interconnecting multiple sensors ofthe array. All of the FETs in each sensor are of a same channel type andimplemented in a single semiconductor region of an array substrate. Acollection of chemFET output signals from all chemFETs of the arrayconstitutes a frame of data. The apparatus further comprises controlcircuitry coupled to the array and configured to generate at least onearray output signal to provide multiple frames of data from the array ata frame rate of at least 20 frames per second.

As an example of the integration of microwell and sensor arrays, FIG. 3shows a composite cross-sectional view of neighboring pixelsillustrating a layer-by-layer view of the pixel fabrication and relativepositions of floating gates and microwells. Three adjacent pixels areshown in cross-section. All of the FET components of the pixel 1051 arefabricated asp-channel FETs in the single n-type well 154. Additionally,in the composite cross-sectional view of FIG. 3 the highly doped p-typeregion 159 is also visible corresponding to the shared drain (D) of theMOSFETs Q2 and Q3. For purposes of illustration, the polysilicon gate166 of the MOSFET Q3 also is visible in FIG. 3. However, for simplicity,the respective sources of the MOSFETs Q2 and Q3 shown in FIG. 2, as wellas the gate of Q2, are not visible in FIG. 3, as they lie along the sameaxis (i.e., perpendicular to the plane of the figure) as the shareddrain. The topmost metal layer 304 corresponds to the ISFETs sensitivearea 178, above which is disposed an analyte-sensitive passivation layer172. The topmost metal layer 304, together with the ISFET polysilicongate 164 and the intervening conductors 306, 308, 312, 316, 320, 326 and338, form the ISFETs floating gate structure 170. However, an electricalconnection to the ISFETs drain is provided by the conductors 340, 328,and 318, coupled to the line 116 ₁ which is formed in the Metal2 layerrather than the Metal3 layer. Additionally, the lines 112 ₁ and 114 ₁also are shown in FIG. 3 as formed in the Metal2 layer rather than theMeta13 layer. The configuration of these lines, as well as the line 118₁, may be further appreciated from the respective images of FIGS. 4Athrough 4L; in particular, it may be observed in FIG. 4F that the line118 ₁, together with the metal conductor 322, is formed in the Metal 1layer, and it may be observed that the lines 112 ₁, 114 ₁ and 116 ₁ areformed in the Metal2 layer, leaving only the jumper 308 of the floatinggate structure 170 in the Meta13 layer shown in FIG. 4J.

Accordingly, by consolidating the signal lines 112 ₁, 114 ₁, 116 ₁, and118 ₁, to the Metal1 and Metal2 layers and thereby increasing thedistance between these signal lines and the topmost layer 304 of thefloating gate structure 170 in the Metal4 layer, parasitic capacitancesin the ISFET may be at least partially mitigated. It should beappreciated that this general concept (e.g., including one or moreintervening metal layers between signal lines and topmost layer of thefloating gate structure) may be implemented in other fabricationprocesses involving greater numbers of metal layers. For example,distance between pixel signal lines and the topmost metal layer may beincreased by adding additional metal layers (more than four total metallayers) in which only jumpers to the topmost metal layer are formed inthe additional metal layers. In particular, a six-metal-layerfabrication process may be employed, in which the signal lines arefabricated using the Metal 1 and Metal2 layers, the topmost metal layerof the floating gate structure is formed in the Metal6 layer, andjumpers to the topmost metal layer are formed in the Metal3, Metal4 andMetal 5 layers, respectively (with associated vias between the metallayers).

In yet another aspect relating to reduced capacitance, a dimension “f”of the topmost metal layer 304 (and thus the ISFET sensitive area 178)may be reduced so as to reduce cross-capacitance between neighboringpixels. As may be observed in FIG. 4 (and as discussed further below inconnection with other embodiments directed to well fabrication above anISFET array), the well 725 may be fabricated so as to have a taperedshape, such that a dimension “g” at the top of the well is smaller thanthe pixel pitch “e” but yet larger than a dimension “f” at the bottom ofthe well. Based on such tapering, the topmost metal layer 304 also maybe designed with the dimension “f” rather than the dimension “g” so asto provide for additional space between the top metal layers ofneighboring pixels. In some illustrative non-limiting implementations,for pixels having a dimension “e” on the order of 9 micrometers thedimension “f” may be on the order of 6 micrometers (as opposed to 7micrometers, as discussed above), and for pixels having a dimension “e”on the order of 5 micrometers the dimension “f” may be on the order of3.5 micrometers.

Detection of hydrogen ions, and other analytes as determined by theinvention, can be carried out using a passivation layer made of siliconnitride (Si₃N₄), silicon oxynitride (Si₂N₂O), silicon oxide (SiO₂),aluminum oxide (Al₂O₃), tantalum pentoxide (Ta₂O₅), tin oxide or stannicoxide (SnO₂), and the like.

When a dielectric layer is added over the floating gate structure of theISFET sensor arrangement, the path from the analyte to the ISFET gatemay be modeled as a series connection of three capacitances: (1) thecapacitance attributable to the above-described charge double layer atthe analyte-dielectric layer interface (labeled C_(DL)), (2) thecapacitance due to the floating gate dielectric layer (C_(FGD)), and (3)the gate oxide capacitance (C_(ox)). (Note that in the text above, thefloating gate dielectric layer is sometimes referred to as a“passivation” layer. Here, we refer more specifically to the layer as afloating gate dielectric layer in order to avoid any suggestion that thematerial composition of the layer is necessarily related to theso-called passivation material(s) often used in CMOS processing (e.g.,PECVD silicon nitride) to coat and protect circuit elements.) The seriescapacitance string extends between the liquid analyte in the wells andthe ISFET gate.

It is well known that capacitances in series form a capacitive voltagedivider. Consequently, only a fraction of the signal voltage, V_(s),generated by or in the analyte, is applied to the gate oxide as thevoltage V_(G) that drives the ISFET. If we define the gate gain asV_(G)/V_(S), one would ideally like to have unity gain—i.e., no signalloss across any of the three capacitances. The value of C_(DL) is afunction of material properties and is typically on the order of about10-40 μF/cm². The gate oxide capacitance is typically a very small valueby comparison. Thus, by making C_(FGD) much greater than the seriescombination of C_(ox) and C_(DL) (for short, C_(FGD)>>C_(ox)), the gategain can be made to approach unity as closely as is practical.

To achieve the relationship C_(FGD)>>C_(ox), one can minimize C_(ox),maximize C_(FGD), or both. Maximization of C_(FGD) can be achieved byusing a thin layer of high dielectric constant material, or byincreasing the area of the floating gate metallization. The capacitanceC_(FGD) is essentially formed by a parallel plate capacitor having thefloating gate dielectric layer as its dielectric. Consequently, for agiven plate (i.e., floating gate metallization) area, the parametersprincipally available for increasing the value of C_(FGD) are (1) thethickness of the dielectric layer and (2) the selection of thedielectric material and, hence, its dielectric constant. The capacitanceof the floating gate dielectric layer varies directly with itsdielectric constant and inversely with its thickness. Thus, a thin,high-dielectric-constant layer would be preferred, to satisfy theobjective of obtaining maximum gate gain.

One candidate for the floating gate dielectric layer material is thepassivation material used by standard CMOS foundry processes. Thestandard (typically, PECVD nitride or, to be more precise, siliconnitride over silicon oxynitride) passivation layer is relatively thickwhen formed (e.g., about 1.3 μm), and typical passivation materials havea limited dielectric constant. A first improvement can be achieved bythinning the passivation layer after formation. This can be accomplishedby etching back the CMOS passivation layer, such as by using anover-etch step during microwell formation, to etch into and consume muchof the nitride passivation layer, leaving a thinner layer, such as alayer only about 200-600 Angstroms thick.

Two approaches have been used for etching a standard CMOS passivationlayer of silicon nitride deposited over silicon oxynitride. A firstapproach is referred to as the “partial etch” technique; it involvesetching away the silicon nitride layer plus approximately half of thesilicon oxynitride layer before depositing the thin-film metal oxidesensing layer. The second approach referred to as the “etch-to-metal”technique, involves etching away all of the silicon nitride and siliconoxynitride layers before depositing the thin-film metal oxide sensinglayer. With an ALD Ta₂O_(s) thin-film sensing layer deposited over a“partial etch,” ISFET gains from about 0.37 to about 0.43 have beenobtained empirically, with sensor sensitivities of about 15.02-17.08mV/pH.

An alternative is to simply deposit a thinner layer of dielectric(passivation) material in the first place, such as the indicated 200-600Angstroms instead of the 1.3 μm of the conventional CMOS passivationprocess. Materials useful for the floating gate dielectric layer aremetal oxides such as tantalum oxide, tungsten oxide, aluminum oxide, andhafnium oxide, though other materials of dielectric constant greaterthan that of the usual silicon nitride passivation material may besubstituted, provided that such material is, or can be rendered,sensitive to the ion of interest. The etch-to-metal approach ispreferred, with the CMOS process' passivation oxide on the floating gatebeing etched completely away prior to depositing the floating gatedielectric material layer. That dielectric layer may be applied directlyon the metal extended ISFET floating gate electrode. This will helpmaximize the value of the capacitance C_(FGD). FIGS. 5A and 5B showsteps using readily available fabrication techniques for generating adielectric layer for high capacitive coupling to array sensor plates.Process steps additionally provide electrical access to bondpadstructures for off-chip communication. Initially wafer (500) from asemiconductor manufacturer is treated to apply material layer (502) fromwhich microwells are formed (in this example, TEOS), after whichmicrowells are formed by etching to the metal of the senor plate (504).Dielectric layer (506) is added, for example, by atomic layerdeposition. In one embodiment, as illustrated in FIG. 5B, dielectriclayer (506) comprises a charge-sensitive layer (512) and an adhesionlayer (514). Using alternative techniques and materials either single ormultipli-component dielectric layers may be formed. Tantalum oxide andaluminum are exemplary charge-sensitive and adhesion layers ofdielectric layer (506). As mentioned above, other materials from whichdielectric layer (506) may be formed include Ta₂O₅ 5, Al₂O₃, HfO₂ orWO₃. In particular, such materials result in a larger signal in responseto pH changes in a sample fluid. Iridium oxide may also be used, e.g. asdescribed in D. O. Wipf et al., “Microscopic Measurement of pH withIridium Oxide Microelectrodes,” Anal. Chem. 2000, 72, 4921-4927, and Y.J. Kim et al., “Configuration for Micro pH Sensor,” Electronics Letters,Vol. 39, No. 21 (Oct. 16, 2003).

Chip Control and Readout Circuitry

A wide variety of on-chip architectures and circuit designs may be usedfor acquiring and processing output signals generated by sensors inarrays of the invention. Several approaches are disclosed in Rothberg etal., U.S. patent publications 2009/0026082 and 2009/0127589, which maybe used with arrays of the present invention. For example, FIG. 6illustrates a block diagram of the sensor array 100 coupled to an arraycontroller 250, according to one inventive embodiment of the presentdisclosure. In various exemplary implementations, the array controller250 may be fabricated as a “stand alone” controller, or as one or morecomputer compatible “cards” forming part of a computer 260 In oneaspect, the functions of the array controller 250 may be controlled bythe computer 260 through an interface block 252 (e.g., serial interface,via USB port or PCI bus, Ethernet connection, etc.). In one embodiment,all or a portion of the array controller 250 is fabricated as one ormore printed circuit boards, and the array 100 is configured to pluginto one of the printed circuit boards, similar to a conventional ICchip (e.g., the array 100 is configured as an ASIC that plugs into achip socket, such as a zero-insertion-force or “ZIF” socket, of aprinted circuit board). In one aspect of such an embodiment, an array100 configured as an ASIC may include one or more pins/terminalconnections dedicated to providing an identification code that may beaccessed/read by the array controller 250 and/or passed on to thecomputer 260. Such an identification code may represent variousattributes of the array 100 (e.g., size, number of pixels, number ofoutput signals, various operating parameters such as supply and/or biasvoltages, etc.) and may be processed to determine correspondingoperating modes, parameters and or signals provided by the arraycontroller 250 to ensure appropriate operation with any of a number ofdifferent types of arrays 100. In one exemplary implementation, an array100 configured as an ASIC may be provided with three pins dedicated toan identification code, and during the manufacturing process the ASICmay be encoded to provide one of three possible voltage states at eachof these three pins (i.e., a tri-state pin coding scheme) to be read bythe array controller 250, thereby providing for unique arrayidentification codes. In another aspect of this embodiment, all orportions of the array controller 250 may be implemented as a fieldprogrammable gate array (FPGA) configured to perform various arraycontroller functions described in further detail below.

Generally, the array controller 250 provides various supply voltages andbias voltages to the array 100, as well as various signals relating torow and column selection, sampling of pixel outputs and dataacquisition. In particular, the array controller 250 reads one or moreanalog output signals (e.g., V_(out1) and V_(out2)) includingmultiplexed respective pixel voltage signals from the array 100 and thendigitizes these respective pixel signals to provide measurement data tothe computer 260, which in turn may store and/or process the data. Insome implementations, the array controller 250 also may be configured toperform or facilitate various array calibration and diagnosticfunctions. Array controller 250 generally provides to the array 100 theanalog supply voltage and ground (VDDA, VSSA), the digital supplyvoltage and ground (VDDD, VSSD), and the buffer output supply voltageand ground (VDDO, VSSO). In one exemplary implementation, each of thesupply voltages VDDA, VDDD and VDDO is approximately 3.3 Volts. Inanother implementation, the supply voltages VDDA, VDDD and VDDO may beas low as approximately 1.8 Volts. As discussed above, in one aspecteach of these power supply voltages is provided to the array 100 viaseparate conducting paths to facilitate noise isolation. In anotheraspect, these supply voltages may originate from respective powersupplies/regulators, or one or more of these supply voltages mayoriginate from a common source in a power supply 258 of the arraycontroller 250. The power supply 258 also may provide the various biasvoltages required for array operation (e.g., VBI, VB2, VB3, VB4, VBO0,V_(BODY)) and the reference voltage VREF used for array diagnostics andcalibration.

In another aspect, the power supply 258 includes one or moredigital-to-analog converters (DACs) that may be controlled by thecomputer 260 to allow any or all of the bias voltages, referencevoltage, and supply voltages to be changed under software control (i.e.,programmable bias settings). For example, a power supply 258 responsiveto computer control (e.g., via software execution) may facilitateadjustment of one or more of the supply voltages (e.g., switchingbetween 3.3 Volts and 1.8 Volts depending on chip type as represented byan identification code), and or adjustment of one or more of the biasvoltages VBI and VB2 for pixel drain current, VB3 for column bus drive,VB4 for column amplifier bandwidth, and VBO0 for column output buffercurrent drive. In some aspects, one or more bias voltages may beadjusted to optimize settling times of signals from enabled pixels.Additionally, the common body voltage V_(BODY) for all ISFETs of thearray may be grounded during an optional post-fabrication UV irradiationtreatment to reduce trapped charge, and then coupled to a higher voltage(e.g., VDDA) during diagnostic analysis, calibration, and normaloperation of the array for measurement/data acquisition. Likewise, thereference voltage VREF may be varied to facilitate a variety ofdiagnostic and calibration functions.

As shown in FIG. 6, the reference electrode 76 which is typicallyemployed in connection with an analyte solution to be measured by thearray 100 may be coupled to the power supply 258 to provide a referencepotential for the pixel output voltages. For example, in oneimplementation the reference electrode 76 may be coupled to a supplyground (e.g., the analog ground VSSA) to provide a reference for thepixel output voltages. In other exemplary implementations, the referenceelectrode voltage may be set by placing a solution/sample of interesthaving a known pH level in proximity to the sensor array 100 andadjusting the reference electrode voltage until the array output signalsV_(out1) and V_(out2) provide pixel voltages at a desired referencelevel, from which subsequent changes in pixel voltages reflect localchanges in pH with respect to the known reference pH level. In general,it should be appreciated that a voltage associated with the referenceelectrode 76 need not necessarily be identical to the reference voltageVREF discussed above (which may be employed for a variety of arraydiagnostic and calibration functions), although in some implementationsthe reference voltage VREF provided by the power supply 258 may be usedto set the voltage of the reference electrode 76.

Regarding data acquisition from the array 100, in one embodiment thearray controller 250 of FIG. 6 may include one or more preamplifiers 253to further buffer one or more output signals (e.g., V_(out1) andV_(out2)) from the sensor array and provide selectable gain. In oneaspect, the array controller 250 may include one preamplifier for eachoutput signal (e.g., two preamplifiers for two analog output signals).In other aspects, the preamplifiers may be configured to accept inputvoltages from 0.0 to 1.8 Volts or 0.0 to 3.3 Volts, may haveprogrammable/computer selectable gains (e.g., 1, 2, 5, 10 and 20) andlow noise outputs (e.g., <10 n V/sqrtHz), and may provide low passfiltering (e.g., bandwidths of 5 MHz and 25 MHz). With respect to noisereduction and increasing signal-to-noise ratio, in one implementation inwhich the array 100 is configured as an ASIC placed in a chip socket ofa printed circuit board containing all or a portion of the arraycontroller 250, filtering capacitors may be employed in proximity to thechip socket (e.g., the underside of a ZIF socket) to facilitate noisereduction. In yet another aspect, the preamplifiers may have aprogrammable/computer selectable offset for input and/or output voltagesignals to set a nominal level for either to a desired range.

The array controller 250 of FIG. 6 also comprises one or moreanalog-to-digital converters 254 (ADCs) to convert the sensor arrayoutput signals V_(out1) and V_(out2) to digital outputs (e.g., 10-bit or12-bit) so as to provide data to the computer 260. In one aspect, oneADC may be employed for each analog output of the sensor array, and eachADC may be coupled to the output of a corresponding preamplifier (ifpreamplifiers are employed in a given implementation). In anotheraspect, the ADC(s) may have a computer-selectable input range (e.g., 50mV, 200 mV, 500 mV, IV) to facilitate compatibility with differentranges of array output signals and/or preamplifier parameters. In yetother aspects, the bandwidth of the ADC(s) may be greater than 60 MHz,and the data acquisition/conversion rate greater than 25 MHz (e.g., ashigh as 100 MHz or greater).

In the embodiment of FIG. 6, ADC acquisition timing and array row andcolumn selection may be controlled by a timing generator 256. Inparticular, the timing generator provides the digital vertical data andclock signals (DV, CV) to control row selection, the digital horizontaldata and clock signals (DH, CH) to control column selection, and thecolumn sample and hold signal COL SH to sample respective pixel voltagesfor an enabled row. The timing generator 256 also provides a samplingclock signal CS to the ADC(s) 254 so as to appropriately sample anddigitize consecutive pixel values in the data stream of a given arrayanalog output signal (e.g., V_(out1) and V_(out2)), as discussed furtherbelow in connection with FIG. 7. In some implementations, the timinggenerator 256 may be implemented by a microprocessor executing code andconfigured as a multichannel digital pattern generator to provideappropriately timed control signals. In one exemplary implementation,the timing generator 256 may be implemented as a field-programmable gatearray (FPGA).

FIG. 7 illustrates an exemplary timing diagram for various array controlsignals, as provided by the timing generator 256, to acquire pixel datafrom the sensor array 100. For purposes of the following discussion, a“frame” is defined as a data set that includes a value (e.g., pixeloutput signal or voltage V_(s)) for each pixel in the array, and a“frame rate” is defined as the rate at which successive frames may beacquired from the array. Thus, the frame rate corresponds essentially toa “pixel sampling rate” for each pixel of the array, as data from anygiven pixel is obtained at the frame rate.

In the example of FIG. 7, an exemplary frame rate of 20 frames/sec ischosen to illustrate operation of the array (i.e., row and columnselection and signal acquisition); however, it should be appreciatedthat arrays and array controllers according to the present disclosureare not limited in this respect, as different frame rates, includinglower frame rates (e.g., 1 to 10 frames/second) or higher frame rates(e.g., 25, 30, 40, 50, 60, 70 to 100 frames/sec., etc.), with arrayshaving the same or higher numbers of pixels, are possible. In someexemplary applications, a data set may be acquired that includes manyframes over several seconds to conduct an experiment on a given analyteor analytes. Several such experiments may be performed in succession, insome cases with pauses in between to allow for data transfer/processingand/or washing of the sensor array ASIC and reagent preparation for asubsequent experiment.

For example, with respect to the method for detecting nucleotideincorporation, appropriate frame rates may be chosen to sufficientlysample the JSFET's output signal. In some exemplary implementations, ahydrogen ion signal may have a full-width at half-maximum (FWHM) on theorder of approximately 1 second to approximately 2.5 seconds, dependingon the number of nucleotide incorporation events. Given these exemplaryvalues, a frame rate (or pixel sampling rate) of 20 Hz is sufficient toreliably resolve the signals in a given pixel's output signal. Again,the frame rates given in this example are provided primarily forpurposes of illustration, and different frame rates may be involved inother implementations.

In one implementation, the array controller 250 controls the array 100to enable rows successively, one at a time. For example, a first row ofpixels is enabled via the row select signal RowSel1 . The enabled pixelsare allowed to settle for some time period, after which the COL SHsignal is asserted briefly to close the sample/hold switch in eachcolumn and store on the column's sample/hold capacitor C_(sh) thevoltage value output by the first pixel in the column. This voltage isthen available as the column output voltage V_(COLj) applied to one ofthe two (odd and even column) array output drivers. The COL SH signal isthen de-asserted, thereby opening the sample/hold switches in eachcolumn and decoupling the column output buffer 111 _(j) from the columnamplifiers 107A and 107B. Shortly thereafter, the second row of pixelsis enabled via the row select signal RowSel2 . During the time period inwhich the second row of pixels is allowed to settle, the column selectsignals are generated two at a time (one odd and one even; odd columnselect signals are applied in succession to the odd output driver, evencolumn select signals are applied in succession to the even outputdriver) to read the column output voltages associated with the firstrow. Thus, while a given row in the array is enabled and settling, theprevious row is being read out, two columns at a time. By staggering rowselection and sampling/readout (e.g., via different vertical andhorizontal clock signals and column sample/hold), and by readingmultiple columns at a time for a given row, a frame of data may beacquired from the array in a significantly streamlined manner.

FIG. 7 illustrates the timing details of the foregoing process for anexemplary frame rate of 20 frames/sec. In a 512×512 array, each row mustbe read out in approximately 98 microseconds, as indicated by thevertical delineations in FIG. 7. Accordingly, the vertical clock signalCV has a period of 98 microseconds (i.e., a clock frequency of over 10kHz), with a new row being enabled on a trailing edge (negativetransition) of the CV signal. The left side of FIG. 7 reflects thebeginning of a new frame cycle, at which point the vertical data signalDV is asserted before a first trailing edge of the CV signal andde-asserted before the next trailing edge of the CV signal. Also,immediately before each trailing edge of the CV signal (i.e., new rowenabled), the COL SH signal is asserted for 2 microseconds, leavingapproximately 50 nanoseconds before the trailing edge of the CV signal.

In FIG. 7, the first occurrence of the COL SH signal is actuallysampling the pixel values of row 512 of the 512×512 array. Thus, uponthe first trailing edge of the CV signal, the first row is enabled andallowed to settle (for approximately 96 microseconds) until the secondoccurrence of the COL SH signal. During this settling time for the firstrow, the pixel values of row 512 are read out via the column selectsignals. Because two column select signals are generated simultaneouslyto read 512 columns, the horizontal clock signal CH must generate 256cycles within this period, each trailing edge of the CH signalgenerating one odd and one even column select signal. As shown in FIG.7, the first trailing edge of the CH signal in a given row is timed tooccur two microseconds after the selection of the row (afterdeactivation of the COL SH signal) to allow for settling of the voltagevalues stored on the sample/hold capacitors C_(sh) and provided by thecolumn output buffers. It should be appreciated however that, in otherimplementations, the time period between the first trailing edge of theCH signal and a trailing edge (i.e., deactivation) of the COL SH signalmay be significantly less than two microseconds, and in some cases assmall as just over 50 nanoseconds. Also for each row, the horizontaldata signal DH is asserted before the first trailing edge of the CHsignal and de-asserted before the next trailing edge of the CH signal.The last two columns are selected before the occurrence of the COL SHsignal which, as discussed above, occurs approximately two microsecondsbefore the next row is enabled. Thus, in the above example, columns areread, two at a time, within a time period of approximately 94microseconds (i.e., 98 microseconds per row, minus two microseconds atthe beginning and end of each row). This results in a data rate for eachof the array output signals V_(out1) and V_(out2) of approximately 2.7MHz.

Noise coupled into the sample fluid by the sensors in every column ofthe array may be present in the output signals of a sensor. When a rowis selected in the array, the drain terminal voltage shared between allof the ISFETs in a column moves up or down (as a necessary requirementof the source-and-drain follower). This changes the gate-to-draincapacitances of all of the unselected ISFETs in the column. In turn,this change in capacitance couples from the gate of every unselectedISFET into the fluid, ultimately manifesting itself as noise in thefluid (i.e., an incorrect charge, one not due to the chemical reactionbeing monitored). That is, any change in the shared drain terminalvoltage can be regarded as injecting noise into the fluid by each andevery unselected ISFET in the column. Hence, if the shared drainterminal voltage of the unselected ISFETs can be kept constant whenselecting a row in the array, this mechanism of coupling noise into thefluid can be reduced or even effectively eliminated. When a row isselected in the array, the source terminal voltage of all of theunselected ISFETs in the column also changes. In turn, that changes thegate-to-source capacitance of all of these ISFETs in the column. Thischange in capacitance couples from the gate of every unselected ISFETinto the fluid, again ultimately manifesting itself as noise in thefluid. That is, any change in the source terminal voltage of anunselected ISFET in the column can be regarded as an injection of noiseinto the fluid. Hence, if the source terminal voltage of the unselectedISFETs can be kept when selecting a row in the array, this mechanism ofcoupling noise into the fluid via can be reduced or even effectivelyeliminated.

A column buffer may be used with some passive pixel designs to alleviatethe ISFET drain problem but not the ISFET source problem. Thus, a columnbuffer most likely is preferable to the above-illustratedsource-and-drain follower. With the illustrated three-transistor passivepixels employing a source-and-drain follower arrangement, there areessentially two sense nodes, the ISFET source and drain terminals, Byconnecting the pixel to a column buffer and grounding the drain terminalof the ISFET, there will be only one sense node: the ISFET sourceterminal. So the drain problem is eliminated.

The above-described readout circuit, which comprises bothsample-and-hold and multiplexer blocks, also has a gain that is lessthan the ideal value of unity. Furthermore, the sample and—hold blockcontributes a significant percentage of the overall chip noise, perhapsmore than 25%. From switched-capacitor theory, the sample-and-hold“kT/C” noise is inversely proportional to capacitance. Hence, bychoosing a larger capacitor, the sample-and-hold noise can be reduced.Another approach to reducing noise is to employ Correlated DoubleSampling (CDS), where a second sample-and-hold and difference circuit isused to cancel out correlated noise. This approach is discussed atgreater length, below.

Correlated Double Sampling (CDS) is a known technique for measuringelectrical values such as voltages or currents that allows for removalof an undesired offset. The output of the sensor is measured twice: oncein a known condition and once in an unknown condition. The valuemeasured from the known condition is then subtracted from the unknowncondition to generate a value with a known relation to the physicalquantity being measured. The challenge here is how to be efficient inimplementing CDS and how to address both correlated noise and theminimization of noise injection into the analyte fluid.

A starting point is the sensor pixel and its readout configuration asexpressed in earlier parts of this application. Referring to FIG. 8A,the basic passive sensor pixel 77A1 is a three transistor arrangement ofan ISFET 77A2 and a pair of row select transistors, 77A3 and 77A4connected to the ISFET source. Transistor 77A3 is connected in turn to acurrent source or sink 77A5. A readout is obtained via transistor 77A4which is connected to the input of sense amplifier 77A6. Adiode-connected transistor 77A7 in series with another amplifier, 77A8,connects in a feedback loop from the output of the sense amplifier tothe drain of the ISFET. The sense amplifier output is captured by asample-and-hold circuit 77A9, which feeds an output amplifier 77A10.

As discussed above, the voltage changes on the ISFET source and draininject noise into the analyte, causing errors in the sensed values. Twoconstructive modifications can reduce the noise level appreciably, asshown in FIG. 8B.

The first change is to alter the signals on the ISFET. The feedback loopto the drain of the ISFET is eliminated and the drain is connected to astable voltage, such as ground. Column buffer 77B is connected to theemitter of transistor.

The second change is to include a circuit to perform CDS on the outputof the column buffer. As mentioned above, CDS requires a first,reference value. This is obtained by connecting the input of columnbuffer 77B1 to a reference voltage via switch 77B2, during a first, orreference phase of a clock, indicated as the “SH” phase. A combined CDSand sample-and-hold circuit 77B3 then double samples the output of thecolumn buffer, obtaining a reference sample and a sensed value, performsa subtraction, and supplies a resulting noise-reduced output value,since the same correlated noise appears in the reference sample and inthe sensor output.

The operation of the CDS and sample-and-hold circuit 77B3 isstraightforward. The circuit operates on a two-phase clock, the firstphase being the SH phase and the second phase being the SHb phase.Typically, the phases will be symmetrical and thus inverted values ofeach other. The reference sample is obtained in the SH phase and placesa charge (and thus a voltage) on capacitor Cin, which is subtracted fromthe output of the column buffer when the clock phase changes.

An alternative embodiment, still with a passive sensor pixel, is shownin FIG. 8C. The sensor pixel in this embodiment is a two-transistorcircuit comprising ISFET whose drain is connected to a fixed supplyvoltage, VSSA. There is no transistor comparable to 77A4, and the pixeloutput is taken from the emitter of transistor 77A3, instead. The CDSand sample-and-hold circuit 77C has been simplified slightly, by theelimination of a feedback loop, but it serves the same function, inconjunction with the charge (voltage) stored on capacitor Cbt, ofsubtracting a reference value on capacitor Cin from the signal suppliedby the sensor pixel.

Microwell Arrays

As discussed elsewhere, for many uses, such as in DNA sequencing, it isdesirable to provide over the array of semiconductor sensors acorresponding array of microwells, each microwell being small enoughpreferably to receive only one DNA-loaded bead, in connection with whichan underlying pixel in the array will provide a corresponding outputsignal.

The use of such a microwell array involves three stages of fabricationand preparation, each of which is discussed separately: (1) creating thearray of microwells to result in a chip having a coat comprising amicrowell array layer; (2) mounting of the coated chip to a fluidicinterface; and in the case of DNA sequencing, (3) loading DNA-loadedbead or beads into the wells. It will be understood, of course, that inother applications, beads may be unnecessary or beads having differentcharacteristics may be employed.

The systems described herein can include an array of microtluidicreaction chambers integrated with a semiconductor comprising an array ofchemFETs. In some embodiments, the invention encompasses such an array.The reaction chambers may, for example, be formed in a glass,dielectric, photodefineable or etchable material. The glass material maybe silicon dioxide.

Various aspects or embodiments of the invention involve an apparatuscomprising an array of chemFET sensors overlayed with an array ofreaction chambers wherein the bottom of a reaction chamber is in contactwith (or capacitively coupled to) a chemFET sensor. In some embodiments,each reaction chamber bottom is in contact with a chemFET sensor, andpreferably with a separate chemFET sensor. In some embodiments, lessthan all reaction chamber bottoms are in contact with a chemFET sensor.In some embodiments, each sensor in the array is in contact with areaction chamber. In other embodiments, less than all sensors are incontact with a reaction chamber. The sensor (and/or reaction chamber)array may be comprised of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40,50, 60, 60, 80, 90, 100, 200, 300, 400, 500, 1000, 10⁴, 10⁵, 10⁶, 10⁷,10⁸, or more chemFET sensors (and/or reaction chambers). As used herein,it is intended that an array that comprises, as an example, 256 sensorsor reaction chambers will contain 256 or more (i.e., at least 256)sensors or reaction chambers. It is further intended that aspects andembodiments described herein that “comprise” elements and/or steps alsofully support and embrace aspects and embodiments that “consist of” or“consist essentially of” such elements and/or steps.

Various aspects and embodiments of the invention involve sensors (and/orreaction chambers) within an array that are spaced apart from each otherat a center-to-center distance or spacing (or “pitch”, as the terms areused interchangeably herein) that is in the range of 1-50 microns, 1-40microns, 1-30 microns, 1-20 microns, 1-10 microns, or 5-10 microns,including equal to or less than about 9 microns, or equal to or lessthan about 5.1 microns, or 1-5 microns including equal to or less thanabout 2.8 microns. The center-to-center distance between adjacentreaction chambers in a reaction chamber array may be about 1-9 microns,or about 2-9 microns, or about 1 microns, about 2 microns, about 3microns, about 4 microns, about 5 microns, about 6 microns, about 7microns, about 8 microns, or about 9 microns.

In some embodiments, the reaction chamber has a volume of equal to orless than about 1 picoliter (pL), including less than 0.5 pL, less than0.1 pL, less than 0.05 pL, less than 0.01 pL, less than 0.005 pL.

The reaction chambers may have a square cross section, for example, attheir base or bottom. Examples include an 8 μm by 8 μm cross section, a4 μm by 4 μm cross section, or a 1.5 μm by 1.5 μm cross section.Alternatively, they may have a rectangular cross section, for example,at their base or bottom. Examples include an 8 μm by 12 μm crosssection, a 4 μm by 6 μm cross section, or a 1.5 μm by 2.25 μm crosssection.

In another exemplary implementation, the invention encompasses a systemcomprising at least one two-dimensional array of reaction chambers,wherein each reaction chamber is coupled to a chemically-sensitive fieldeffect transistor (“chemFET”) and each reaction chamber is no greaterthan 10 μm³ (i.e., 1 pL) in volume. Preferably, each reaction chamber isno greater than 0.34 pL, and more preferably no greater than 0.096 pL oreven 0.012 pL in volume. A reaction chamber can optionally be 2², 3²,4², 5², 6², 7², 8², 9², or 10² square microns in cross-sectional area atthe top. Preferably, the array has at least 10², 10³, 10⁴, 10⁹, 10⁶,10⁷, 10⁸, 10⁹, or more reaction chambers. The reaction chambers may becapacitively coupled to the chemFETs, and preferably are capacitivelycoupled to the chemFETs. Such systems may be used for high-throughputsequencing of nucleic acids.

In some embodiments, the reaction chamber array (or equivalently,microwell array) comprises 10², 10³, 10⁴, 10⁹, 10⁶, 10⁷ microwells orreaction chambers. In some embodiments, individual reaction chambers inthe reaction chamber array are in contact with or capacitively coupledto at least one chemFET. In one embodiment, a reaction chamber of anarray is in contact with or capacitively coupled to one chemFET or oneISFET. In some embodiments, the chemFET array may optionally comprise10², 10³, 10⁴, 10⁵, 10⁶, 10⁷ chemFETs.

In these and in other aspects and embodiments, the chemFET or ISFETarrays may comprise 256 or more chemFETs or ISFETs. The chemFETs orISFETs of such arrays may have a center-to-center spacing (betweenadjacent chemFETs or ISFETs) of 1-10 microns. In some embodiments, thecenter-to-center spacing is about 9 microns, about 8 microns, about 7microns, about 6 microns, about 5 microns, about 4 microns, about 3microns, about 2 microns or about 1 micron. In particular embodiments,the center-to-center spacing is about 5.1 microns or about 2.8 microns.

In some embodiments, the bead is in a reaction chamber, and optionallythe only bead in the reaction chamber. In some embodiments, the reactionchamber is in contact with or capacitively coupled to an ISFET. In someembodiments, the ISFET is in an ISFET array In some embodiments, thebead has a diameter of less than 6 microns, less than 3 microns, orabout 1 micron. The bead may have a diameter of about 1 micron up toabout 7 microns, or about 1 micron up to about 3 microns.

In some embodiments, the reaction chambers have a center-to-centerdistance of about 1 micron to about 10 microns. In some embodiments, thereaction chamber array comprises 10², 10³, 10⁴, 10⁵, 10⁶ or 10⁷ reactionchambers.

In accordance with the invention, a dielectric layer on a gate of anISFET is part of the ISFET. It is recognized that the charge in thereaction chamber builds up on one side of the dielectric and forms oneplate of a capacitor and which has as its second plate the floating gatemetal layer; thus, a reaction chamber is referred to as beingcapacitively coupled to the ISFET.

In some embodiments, the ISFET is in an ISFET array. The ISFET array maycomprise 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷ ISFETS.

In some embodiments, the template nucleic acid is in a reaction chamberin contact with or capacitively coupled to the ISFET. In someembodiments, the reaction chamber is in a reaction chamber array. Insome embodiments, the reaction chamber array comprises 10², 10³, 10⁴,10⁵, 10⁶ or 10⁷ reaction chambers.

The microwells may vary in size between arrays. The size of thesemicrowells may be described in terms of a width (or diameter) to heightratio. In some embodiments, this ratio is I:1 to 2:1.5. The bead to wellsize (e.g., the bead diameter to well width, diameter, or height) ispreferably in the range of 0.6-0.8.

The microwell size may be described in terms of cross section. The crosssection may refer to a “slice” parallel to the depth (or height) of thewell, or it may be a slice perpendicular to the depth (or height) of thewell. The microwells may be square in cross-section, but they are not solimited. The dimensions at the bottom of a microwell (i.e., in a crosssection that is perpendicular to the depth of the well) may be 1.5 μm by1.5 μm, or it may be 1.5 μm by 2 μm. Suitable diameters include but arenot limited to at or about 100 μm, 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 J.tm, 35 μm, 30 μm, 25 μm, 20μm, 15 μm, 10 J.tm, 9 μm, 8 μm, 7 μm, 6 J.tm, 5 J.tm, 4 μm, 3 μm, 2 μm,1 μm or less. In some particular embodiments, the diameters may be at orabout 44 J.tm, 32 J.tm, 85 μm, 4 μm, or 1.5 μm. Suitable heights includebut are not limited to at or about 100 μm, 95 μm, 90 μm, 85 μm, 80 μm,75 J.tm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm,25 μm, 20 μm, 15 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2μm, 1 μm or less. In some particular embodiments, the heights may be ator about 55 μm, 48 μm, 32 μm, 12 μm, 8 μm, 6 μm, 4 μm, 2.25 μm, 1.5 μm,or less. Various embodiments of the invention contemplate thecombination of any of these diameters with any of these heights. Instill other embodiments, the reaction well dimensions may be (diameterin μm by height in μm) 44 by 55, 32 by 32, 32 by 48, 8 by 8, 8 by 12, 4by 4, 4 by 6, 1.5 by 1.5, or 1.5 by 2.25.

The reaction well volume may range (between arrays, and preferably notwithin a single array) based on the well dimensions. This volume may beat or about 100 picoliter (pL), 90, 80, 70, 60, 50, 40, 30, 20, 10, orfewer pL. In important embodiments, the well volume is less than 1 pL,including equal to or less than 0.5 pL, equal to or less than 0.1 pL,equal to or less than 0.05 pL, equal to or less than 0.01 pL, equal toor less than 0.005 pL, or equal to or less than 0.001 pL. The volume maybe 0.001 to 0.9 pL, 0.001 to 0.5 pL, 0.001 to 0.1 pL, 0.001 to 0.05 pL,or 0.005 to 0.05 pL. In particular embodiments, the well volume is 75pL, 34 pL, 23 pL, 0.54 pL, 0.36 pL, 0.07 pL, 0.045 pL, 0.0024 pL, or0.004 pL. In some embodiments, each reaction chamber is no greater thanabout 0.39 pL in volume and about 49 μm² surface aperture, and morepreferably has an aperture no greater than about 16 μm² and volume nogreater than about 0.064 pL.

Thus, it is to be understood that various aspects and embodiments of theinvention relate generally to large scale FET arrays for measuring oneor more analytes or for measuring charge bound to the chemFET surface.It will be appreciated that chemFETs and more particularly ISFETs may beused to detect analytes and/or charge. An ISFET, as discussed above, isa particular type of chemFET that is configured for ion detection suchas hydrogen ion (or proton) detection. Other types of chemFETscontemplated by the present disclosure include enzyme FETs (EnFETs)which employ enzymes to detect analytes. It should be appreciated,however, that the present disclosure is not limited to ISFETs andEnFETs, but more generally relates to any FET that is configured forsome type of chemical sensitivity. As used herein, chemical sensitivitybroadly encompasses sensitivity to any molecule of interest, includingwithout limitation organic, inorganic, naturally occurring,non-naturally occurring, chemical and biological compounds, such asions, small molecules, polymers such as nucleic acids, proteins,peptides, polysaccharides, and the like.

In some embodiments, the invention encompasses a sequencing apparatuscomprising a dielectric layer overlying a chemFET, the dielectric layerhaving a recess laterally centered atop the chemFET. Preferably, thedielectric layer is formed of silicon dioxide.

After the semiconductor structures, as shown, are formed, the microwellstructure is applied to the die. That is, the microwell structure can beformed right on the die or it may be formed separately and then mountedonto the die, either approach being acceptable. To form the microwellstructure on the die, various processes may be used. For example, theentire die may be spin-coated with, for example, a negative photoresistsuch as Microchem's SU-8 2015 or a positive resist/polyimide such as HDMicrosystems HD8820, to the desired height of the microwells. Thedesired height of the wells (e.g., about 4-12 μm in the example of onepixel per well, though not so limited as a general matter) in thephotoresist layer(s) can be achieved by spinning the appropriate resistat predetermined rates (which can be found by reference to theliterature and manufacturer specifications, or empirically), in one ormore layers. (Well height typically may be selected in correspondencewith the lateral dimension of the sensor pixel, preferably for a nominal1:1-1.5:1 aspect ratio, height:width or diameter. Based onsignal-to-noise considerations, there is a relationship betweendimensions and the required data sampling rates to achieve a desiredlevel of performance. Thus there are a number of factors that will gointo selecting optimum parameters for a given application.)Alternatively, multiple layers of different photoresists may be appliedor another form of dielectric material may be deposited. Various typesof chemical vapor deposition may also be used to build up a layer ofmaterials suitable for microwell formation therein.

Once the photoresist layer (the singular form “layer” is used toencompass multiple layers in the aggregate, as well) is in place, theindividual wells (typically mapped to have either one or four ISFETsensors per well) may be generated by placing a mask (e.g., of chromium)over the resist-coated die and exposing the resist to cross-linking(typically UV) radiation. All resist exposed to the radiation (i.e.,where the mask does not block the radiation) becomes cross-linked and asa result will form a permanent plastic layer bonded to the surface ofthe chip (die). Unreacted resist (i.e., resist in areas which are notexposed, due to the mask blocking the light from reaching the resist andpreventing cross-linking) is removed by washing the chip in a suitablesolvent (i.e., developer) such as propyleneglycolmethylethylacetate(PGMEA) or other appropriate solvent. The resultant structure definesthe walls of the microwell array.

For example, contact lithography of various resolutions and with variousetchants and developers may be employed. Both organic and inorganicmaterials may be used for the layer(s) in which the microwells areformed. The layer(s) may be etched on a chip having a dielectric layerover the pixel structures in the sensor array, such as a passivationlayer, or the layer(s) may be formed separately and then applied overthe sensor array. The specific choice or processes will depend onfactors such as array size, well size, the fabrication facility that isavailable, acceptable costs, and the like.

Among the various organic materials which may be used in someembodiments to form the microwelllayer(s) are the above-mentioned SU-8type of negative-acting photoresist, a conventional positive-actingphotoresist and a positive-acting photodefineable polyimide. Each hasits virtues and its drawbacks, well known to those familiar with thephotolithographic art.

Naturally, in a production environment, modifications will beappropriate.

Contact lithography has its limitations and it may not be the productionmethod of choice to produce the highest densities of wells—i.e., it mayimpose a higher than desired minimum pitch limit in the lateraldirections. Other techniques, such as a deep UV step-and-repeat process,are capable of providing higher resolution lithography and can be usedto produce small pitches and possibly smaller well diameters. Of course,for different desired specifications (e.g., numbers of sensors and wellsper chip), different techniques may prove optimal. And pragmaticfactors, such as the fabrication processes available to a manufacturer,may motivate the use of a specific fabrication method. While novelmethods are discussed, various aspects of the invention are limited touse of these novel methods.

Preferably the CMOS wafer with the ISFET array will be planarized afterthe final metallization process. A chemical mechanical dielectricplanarization prior to the silicon nitride passivation is suitable. Thiswill allow subsequent lithographic steps to be done on very flatsurfaces which are free of back-end CMOS topography.

By utilizing deep-UV step-and-repeat lithography systems, it is possibleto resolve small features with superior resolution, registration, andrepeatability. However, the high resolution and large numerical aperture(NA) of these systems precludes their having a large depth of focus. Assuch, it may be necessary, when using such a fabrication system, to usethinner photodefinable spin-on layers (i.e., resists on the order of 1-2μm rather than the thicker layers used in contact lithography) topattern transfer and then etch microwell features to underlying layer orlayers. High resolution lithography can then be used to pattern themicrowell features and conventional SiO₂ etch chemistries can beused—one each for the bondpad areas and then the microwell areas havingselective etch stops; the etch stops then can be on aluminum bondpadsand silicon nitride passivation (or the like), respectively.Alternatively, other suitable substitute pattern transfer and etchprocesses can be employed to render microwells of inorganic materials.

Another approach is to form the microwell structure in an organicmaterial. For example, a dual-resist “soft-mask” process may beemployed, whereby a thin high-resolution deep UV resist is used on topof a thicker organic material (e.g., cured polyimide or opposite-actingresist). The top resist layer is patterned. The pattern can betransferred using an oxygen plasma reactive ion etch process. Thisprocess sequence is sometimes referred to as the “portable conformablemask” (PCM) technique. See B. J. Lin et al., “Practicing the Novolacdeep-UV portable conformable masking technique”, Journal of VacuumScience and Technology 19, No. 4, 1313-1319 (1981); and A. Cooper etal., “Optimization of a photosensitive spin-on dielectric process forcopper inductor coil and interconnect protection in RF SoC devices.

Alternatively a “drill-focusing” technique may be employed, wherebyseveral sequential step-and-repeat exposures are done at different focaldepths to compensate for the limited depth of focus (DOE) ofhigh-resolution steppers when patterning thick resist layers. Thistechnique depends on the stepper NA and DOF as well as the contrastproperties of the resist material.

Thus, microwells can be fabricated by any high aspect ratiophoto-definable or etchable thin-film process, that can providerequisite thickness (e.g., about 4-10 μm). Among the materials believedto be suitable are photosensitive polymers, deposited silicon dioxide,nonphotosensitive polymer which can be etched using, for example, plasmaetching processes, etc. In the silicon dioxide family, TEOS and silanenitrous oxide (SILOX) appear suitable. The final structures are similarbut the various materials present differing surface compositions thatmay cause the target biology or chemistry to react differently.

When the microwell layer is formed, it may be necessary to provide anetch stop layer so that the etching process does not proceed furtherthan desired. For example, there may be an underlying layer to bepreserved, such as a low-K dielectric. The etch stop material should beselected according to the application. SiC and SiN materials may besuitable, but that is not meant to indicate that other materials may notbe employed, instead. These etch-stop materials can also serve toenhance the surface chemistry which drives the ISFET sensor sensitivity,by choosing the etch-stop material to have an appropriate point of zerocharge (PZC). Various metal oxides may be suitable addition to silicondioxide and silicon nitride.

The PZCs for various metal oxides may be found in various texts, such as“Metal Oxides-Chemistry and Applications” by J. Fierro. We have learnedthat Ta20s may be preferred as an etch stop over Al₂O₃ because the PZCof Al₂O₃ is right at the pH being used (i.e., about 8.8) and, hence,right at the point of zero charge. In addition Ta₂O₅ has a highersensitivity to pH (i.e., m V/pH), another important factor in the sensorperformance. Optimizing these parameters may require judicious selectionof passivation surface materials.

Using thin metal oxide materials for this purpose (i.e., as an etch stoplayer) is difficult due to the fact of their being so thinly deposited(typically 200-SOOA). A post-microwell fabrication metal oxidedeposition technique may allow placement of appropriate PZC metal oxidefilms at the bottom of the high aspect ratio microwells.

Electron-beam depositions of (a) reactively sputtered tantalum oxide,(b) nonreactive stoichiometric tantalum oxide, (c) tungsten oxide, or(d) Vanadium oxide may prove to have superior “down-in-well” coveragedue to the superior directionality of the deposition process.

The array typically comprises at least 100 microfluidic wells, each ofwhich is coupled to one or more chemFET sensors. Preferably, the wellsare formed in at least one of a glass (e.g., SiO₂), a polymericmaterial, a photodefinable material or a reactively ion etchable thinfilm material. Preferably, the wells have a width to height ratio lessthan about 1:1. Preferably the sensor is a field effect transistor, andmore preferably a chemFET. The chemFET may optionally be coupled to aPPi receptor. Preferably, each of the chemFETs occupies an area of thearray that is 10² microns or less.

In some embodiments, the invention encompasses a sequencing devicecomprising a semiconductor wafer device coupled to a dielectric layersuch as a glass (e.g., SiO₂), polymeric, photodefinable or reactive ionetchable material in which reaction chambers are formed. Typically, theglass, dielectric, polymeric, photodefinable or reactive ion etchablematerial is integrated with the semiconductor wafer layer. In someinstances, the glass, polymeric, photodefinable or reactive ion etchablelayer is non-crystalline. In some instances, the glass may be SiO₂. Thedevice can optionally further comprise a fluid delivery module of asuitable material such as a polymeric material, preferably an injectionmoldable material. More preferably, the polymeric layer ispolycarbonate.

In some embodiments, the invention encompasses a method formanufacturing a sequencing device comprising: using photolithography,generating wells in a glass, dielectric, photodefinable or reactivelyion etchable material on top of an array of transistors.

Yet another alternative when a CMOS or similar fabrication process isused for array fabrication is to form the microwells directly using theCMOS materials. That is, the CMOS top metallization layer forming thefloating gates of the ISFET array usually is coated with a passivationlayer that is about 1.3 μm thick. Microwells 1.3 μm deep can be formedby etching away the passivation material. For example, microwells havinga 1:1 aspect ratio may be formed, 1.3 μm deep and 1.3 μm across at theirtops. Modeling indicates that as the well size is reduced, in fact, theDNA concentration, and hence the SNR, increases. So, other factors beingequal, such small wells may prove desirable.

Flow Cells and Fluidics System

A complete system for using the sensor array will include suitable fluidsources, valving and a controller for operating the valving to lowreagents and washes over the microarray or sensor array, depending onthe application. These elements are readily assembled from off-the-shelfcomponents, with and the controller may readily be programmed to performa desired experiment.

It should be understood that the readout at the chemFET may be currentor voltage (and change thereof) and that any particular reference toeither readout is intended for simplicity and not to the exclusion ofthe other readout. Therefore any reference in the following text toeither current or voltage detection at the chemFET should be understoodto contemplate and apply equally to the other readout as well. Inimportant embodiments, the readout reflects a rapid, transient change inconcentration of an analyte. The concentration of more than one analytemay be detected at different times. In some instances, such measurementsare to be contrasted with methods that focus on steady stateconcentration measurements.

The process of using the assembly of an array of sensors on a chipcombined with an array of microwells to sequence the DNA in a sample isreferred to as an “experiment.” Executing an experiment requires loadingthe wells with the DNA-bound beads and the flowing of several differentfluid solutions (i.e., reagents and washes) across the wells. A fluiddelivery system (e.g., valves, conduits, pressure source(s), etc.)coupled with a fluidic interface is needed which flows the varioussolutions across the wells in a controlled even flow with acceptablysmall dead volumes and small cross contamination between sequentialsolutions. Ideally, the fluidic interface to the chip (sometimesreferred to as a “flow cell”) would cause the fluid to reach allmicrowells at the same time. To maximize array speed, it is necessarythat the array outputs be available at as close to the same time aspossible. The ideal clearly is not possible, but it is desirable tominimize the differentials, or skews, of the arrival times of anintroduced fluid, at the various wells, in order to maximize the overallspeed of acquisition of all the signals from the array.

Flow cell designs of many configurations are possible; thus the systemand methods presented herein are not dependent on use of a specific flowcell configuration. It is desirable, though, that a suitable flow cellsubstantially conform to the following set of objectives:

-   -   have connections suitable for interconnecting with a fluidics        delivery system—e.g., via appropriately-sized tubing;    -   have appropriate head space above wells;    -   minimize dead volumes encountered by fluids;    -   minimize small spaces in contact with liquid but not quickly        swept clean by flow of a wash fluid through the flow cell (to        minimize cross contamination);    -   be configured to achieve uniform transit time of the flow over        the array;    -   generate or propagate minimal bubbles in the flow over the        wells;    -   be adaptable to placement of a removable reference electrode        inside or as close to the flow chamber as possible;    -   facilitate easy loading of beads;    -   be manufacturable at acceptable cost; and    -   be easily assembled and attached to the chip package.

Satisfaction of these criteria so far as possible will contribute tosystem performance positively. For example, minimization of bubbles isimportant so that signals from the array truly indicate the reaction ina well rather than being spurious noise.

Each of several example designs will be discussed, meeting thesecriteria in differing ways and degrees. In each instance, one typicallymay choose to implement the design in one of two ways: either byattaching the flow cell to a frame and gluing the frame (or otherwiseattaching it) to the chip or by integrating the frame into the flow cellstructure and attaching this unified assembly to the chip. Further,designs may be categorized by the way the reference electrode isintegrated into the arrangement. Depending on the design, the referenceelectrode may be integrated into the flow cell (e.g., form part of theceiling of the flow chamber) or be in the flow path (typically to theoutlet or downstream side of the flow path, after the sensor array).

An example of a suitable experiment apparatus 3410 incorporating such afluidic interface is shown in FIG. 9, the manufacture and constructionof which will be discussed in greater detail below. The apparatuscomprises a semiconductor chip 3412 (indicated generally, though hidden)on or in which the arrays of wells and sensors are formed, and afluidics assembly 3414 on top of the chip and delivering the sample tothe chip for reading. The fluidics assembly includes a portion 3416 forintroducing fluid containing the sample, a portion 3418 for allowing thefluid to be piped out, and a flow chamber portion 3420 for allowing thefluid to flow from inlet to outlet and along the way interact with thematerial in the wells. Those three portions are unified by an interfacecomprising a glass slide 3422 (e.g., Erie Microarray Cat #C22-5128-M20from Erie Scientific Company, Portsmouth, N.H., cut in thirds, each tobe of size about 25 mm×25 mm).

Mounted on the top face of the glass slide are two fittings, 3424 and3426, such as nanoport fittings Part #N-333 from Upchurch Scientific ofOak Harbor, Wash. One port (e.g., 3424) serves as an inlet deliveringliquids from the pumpinglvalving system described below but not shownhere. The second port (e.g., 3426) is the outlet which pipes the liquidsto waste. Each port connects to a conduit 3428, 3432 such as flexibletubing of appropriate inner diameter. The nanoports are mounted suchthat the tubing can penetrate corresponding holes in the glass slide.The tube apertures should be flush with the bottom surface of the slide.

On the bottom of the glass slide, flow chamber 3420 may comprise variousstructures for promoting a substantially laminar flow across themicrowell array. For example, a series of microfluidic channels fanningout from the inlet pipe to the edge of the flow chamber may be patternedby contact lithography using positive photoresists such as SU-8photoresist from MicroChem Corp. of Newton, Mass. Other structures willbe discussed below.

The chip 3412 will in turn be mounted to a carrier 3430, for packagingand connection to connector pins 3432.

Achieving a uniform flow front and eliminating problematic flow pathareas is desirable for a number of reasons. One reason is that very fasttransition of fluid interfaces within the system's flow cell is desiredfor many applications, particularly gene sequencing. In other words, anincoming fluid must completely displace the previous fluid in a shortperiod of time. Uneven fluid velocities and diffusion within the flowcell, as well as problematic flow paths, can compete with thisrequirement. Simple flow through a conduit of rectangular cross sectioncan exhibit considerable disparity of fluid velocity from regions nearthe center of the flow volume to those adjacent the sidewalls, onesidewall being the top surface of the microwell layer and the fluid inthe wells. Such disparity leads to spatially and temporally largeconcentration gradients between the two traveling fluids. Further,bubbles are likely to be trapped or created in stagnant areas like sharpcorners interior the flow cell. (The surface energy (hydrophilic vs.hydrophobic) can significantly affect bubble retention. Avoidance ofsurface contamination during processing and use of a surface treatmentto create a more hydrophilic surface should be considered if theas-molded surface is too hydrophobic.) Of course, the physicalarrangement of the flow chamber is probably the factor which mostinfluences the degree of uniformity achievable for the flow front.

In all cases, attention should be given to assuring a thorough washingof the entire flow chamber, along with the microwells, between reagentcycles. Flow disturbances may exacerbate the challenge of fully cleaningout the flow chamber.

Flow disturbances may also induce or multiply bubbles in the fluid. Abubble may prevent the fluid from reaching a microwell, or delay itsintroduction to the microwell, introducing error into the microwellreading or making the output from that microwell useless in theprocessing of outputs from the array. Thus, care should be taken inselecting configurations and dimensions for the flow disruptor elementsto manage these potential adverse factors. For example, a tradeoff maybe made between the heights of the disruptor elements and the velocityprofile change that is desired.

The flow cell, as stated elsewhere, may be fabricated of many differentmaterials. Injection molded polycarbonate appears to be quite suitable.A conductive metal (e.g., gold) may be deposited using an adhesion layer(e.g., chrome) to the underside of the flow cell roof (the ceiling ofthe flow chamber). Appropriate low-temperature thin-film depositiontechniques preferably are employed in the deposition of the metalreference electrode due to the materials (e.g., polycarbonate) and largestep coverage topography at the bottom-side of the fluidic cell (i.e.,the frame surround of ISFET array). One possible approach would be touse electron-beam evaporation in a planetary system.

Once assembly is complete—conductive epoxy (e.g., Epo-Tek H20E orsimilar) may be dispensed on the seal ring with the flow cell aligned,placed, pressed and cured—the ISFET flow cell is ready for operationwith the reference potential being applied to the assigned pin of thepackage.

In some embodiments, the invention encompasses an apparatus fordetection of pH comprising a laminar fluid flow system. Preferably, theapparatus is used for sequencing a plurality of nucleic acid templatespresent in an array.

The apparatus typically includes a fluidics assembly comprising a membercomprising one or more apertures for non-mechanically directing a fluidto flow to an array of at least 100 K (100 thousand), 500 K (500thousand), or 1 M (1 million) microfluidic reaction chambers such thatthe fluid reaches all of the microfluidic reaction chambers at the sametime or substantially the same time. Typically, the fluid flow isparallel to the sensor surface. Typically, the assembly has a Reynoldsnumber of less than 1000, 500, 200, 100, 50, 20, or 10. Preferably, themember further comprises a first aperture for directing fluid towardsthe sensor array and a second aperture for directing fluid away from thesensor array.

In some embodiments, the invention encompasses a method for directing afluid to a sensor array comprising: providing a fluidics assemblycomprising an aperture fluidly coupling a fluid source to the sensorarray; and non-mechanically directing a fluid to the sensor array. By“non-mechanically” it is meant that the fluid is moved under pressurefrom a gaseous pressure source, as opposed to a mechanical pump.

In some embodiments, the invention encompasses an array of wells, eachof which is coupled to a lid having an inlet port and an outlet port anda fluid delivery system for delivering and removing fluid from saidinlet and outlet ports non-mechanically.

In some embodiments, the invention encompasses a method for sequencing abiological polymer such as a nucleic acid utilizing the above-describedapparatus, comprising: directing a fluid comprising a monomer to anarray of reaction chambers wherein the fluid has a fluid flow Reynoldsnumber of at most 2000, 1000, 200, 100, 50, or 20. The method mayoptionally further comprise detecting a pH or a change in pH from eachsaid reaction chamber. This is typically detected by ion diffusion tothe sensor surface. There are various other ways of providing a fluidicsassembly for delivering an appropriate fluid flow across the microwelland sensor array assembly, and the forgoing examples are thus notintended to be exhaustive.

pH-Based Nucleic Acid Sequencing

Apparatus of the invention may be adapted to detecting hydrogen ionsreleased by nucleotide incorporation, which detection process isdisclosed as a DNA sequencing method in Rothberg et al., U.S. patentpublications 2009/0026082 and 2009/0127589. It is important in these andvarious other aspects to detect as many released hydrogen ions aspossible in order to achieve as high a signal (and/or a signal to noiseratio) as possible. Strategies for increasing the number of releasedprotons that are ultimately detected by the chemFET surface includewithout limitation limiting interaction of released protons withreactive groups in the well, choosing a material from which tomanufacture the well in the first instance that is relatively inert toprotons, preventing released protons from exiting the well prior todetection at the chemFET, and increasing the copy number of templatesper well (in order to amplify the signal from each nucleotideincorporation), among others.

In one aspect, the invention provides arrays and devices with reducedbuffering capacity for monitoring and/or measuring hydrogen ion changes(or pH changes) more accurately. As an example, the invention providesapparatus and devices for monitoring pH changes in polymerase extensionreactions in an environment with no or limited buffering capacity.Examples of a reduced buffering environment include environments thatlack pH buffering components in the sample fluid and/or reactionmixtures; environments in which surfaces of array components in contactwith sample fluid and/or reaction mixtures have little or no bufferingcapacity; and environments in which pH changes on the order of 0.01,0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6. 0.7, 0.8, 0.9, or 1.0 pH units aredetectable for example via a chemFET and more particularly an ISFET asdescribed herein.

The buffering inhibitor may also be a phospholipid. The phospholipidsmay be naturally occurring or non-naturally occurring phospholipids.Examples of phospholipids to be used as buffering inhibitors include butare not limited to phosphatidylcholine, hosphatidylethanolamine,phosphatidylglycerol, and phosphatidylserine. In some embodiments,phospholipids may be coated on the chemFET surface (or reaction chambersurface). Such coating may be covalent or noncovalent. In otherembodiments, the phospholipids exist in solution.

Some instances of the invention employ an environment, including areaction solution, that is minimally buffered, if at all. Buffering canbe contributed by the components of the solution or by the solidsupports in contact with such solution. A solution having no or lowbuffering capacity (or activity) is one in which changes in hydrogen ionconcentration on the order of at least about +/−0.005 pH units, at leastabout +/−0.01, at least about +/−0.015, at least about +/−0.02, at leastabout +/−0.03, at least about +/−0.04, at least about +/−0.05, at leastabout +/−0.10, at least about +/−0.15, at least about +/−0.20, at leastabout +/−0.25, at least about +/−0.30, at least about +/−0.35, at leastabout +/−0.45, at least about +/−0.50, or more are detectable (e.g.,using the chemFET sensors described herein). In some embodiments, the pHchange per nucleotide incorporation is on the order of about 0.005. Insome embodiments, the pH change per nucleotide incorporation is adecrease in pH. Reaction solutions that have no or low bufferingcapacity may contain no or very low concentrations of buffer, or may useweak buffers.

Concatemerized Templates

Increasing the number of templates or primers (i.e., copy number)results in a greater number of nucleotide incorporations per sensorand/or per reaction chamber, thereby leading to a higher signal and thussignal to noise ratio. Copy number can be increased for example by usingtemplates that are concatemers (i.e., nucleic acids comprising multiple,tandemly arranged, copies of the nucleic acid to be sequenced), byincreasing the number of nucleic acids on or in beads up to andincluding saturating such beads, and by attaching templates or primersto beads or to the sensor surface in ways that reduce steric hindranceand/or ensure template attachment (e.g., by covalently attachingtemplates), among other things. Concatemer templates may be immobilizedon or in beads or on other solid supports such as the sensor surface,although in some embodiments concatemers templates may be present in areaction chamber without immobilization. For example, the templates (orcomplexes comprising templates and primers) may be covalently ornon-covalently attached to the chemFET surface and their sequencing mayinvolve detection of released hydrogen ions and/or addition of negativecharge to the chemFET surface upon a nucleotide incorporation event. Thelatter detection scheme may be performed in a buffered environment orsolution (i.e., any changes in pH will not be detected by the chemFETand thus such changes will not interfere with detection of negativecharge addition to the chemFET surface).

RCA or CCR amplification methods generate concatemers of templatenucleic acids that comprise tens, hundreds, thousands or more tandemlyarranged copies of the template. Such concatemers may still be referredto herein as template nucleic acids, although they may contain multiplecopies of starting template nucleic acids. In some embodiments, they mayalso be preferred to as amplified template nucleic acids. Alternatively,they may be referred to herein as comprising multiple copies of targetnucleic acid fragment. Concatemers may contain 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, or more copies ofthe starting nucleic acid. They may contain 10-10², 10²-10³, 10³-10⁴,10³-10⁵, or more copies of the starting nucleic acid. Concatemersgenerated using these or other methods (such as for example DNAnanoballs) can be used in the sequencing-by-synthesis methods describedherein. The concatemers may be generated in vitro apart from the arrayand then placed into reaction chambers of the array or they may begenerated in the reaction chambers. One or more inside walls of thereaction chamber may be treated to enhance attachment and retention ofthe concatemers, although this is not required. In some embodiments ofthe invention, if the concatemers are attached to an inside wall of thereaction chamber, such as the chemFET surface, then nucleotideincorporation at least in the context of a sequencing-by-synthesisreaction may be detected by a change in charge at the chemFET surface,as an alternative to or in addition to the detection of releasedhydrogen ions as discussed herein. If the concatemers are deposited ontoa chemFET surface and/or into a reaction chamber,sequencing-by-synthesis can occur through detection of released hydrogenions as discussed herein. The invention embraces the use of otherapproaches for generating concatemerized templates. One such approach isa PCR described by Stemmer et al. in U.S. Pat. No. 5,834,252, and thedescription of this approach is incorporated by reference herein.

Important aspects of the invention contemplate sequencing a plurality ofdifferent template nucleic acids simultaneously. This may beaccomplished using the sensor arrays described herein. In oneembodiment, the sensor arrays are overlayed (and/or integral with) anarray of microwells (or reaction chambers or wells, as those terms areused interchangeably herein), with the proviso that there be at leastone sensor per microwell. Present in a plurality of microwells is apopulation of identical copies of a template nucleic acid. There is norequirement that any two microwells carry identical template nucleicacids, although in some instances such templates may share overlappingsequence. Thus, each microwell comprises a plurality of identical copiesof a template nucleic acid, and the templates between microwells may bedifferent.

It is to be understood therefore that the invention contemplates asequencing apparatus for sequencing unlabeled nucleic acid acids,optionally using unlabeled nucleotides, without optical detection andcomprising an array of at least 100 reaction chambers. In someembodiments, the array comprises 10³, 10⁴, 10⁵, 10⁶, 10⁷ or morereaction chambers. The pitch (or center-to-center distance betweenadjacent reaction chambers) is on the order of about 1-10 microns,including 1-9 microns, 1-8 microns, 1-7 microns, 1-6 microns, 1-5microns, 1-4 microns, 1-3 microns, or 1-2 microns.

In various aspects and embodiments of the invention, the nucleic acidloaded beads, of which there may be tens, hundreds, thousands, or more,first enter the flow cell and then individual beads enter individualwells. The beads may enter the wells passively or otherwise. Forexample, the beads may enter the wells through gravity without anyapplied external force. The beads may enter the wells through an appliedexternal force including but not limited to a magnetic force or acentrifugal force. In some embodiments, if an external force is applied,it is applied in a direction that is parallel to the well height/depthrather than transverse to the well height/depth, with the aim being to“capture” as many beads as possible. Preferably, the wells (or wellarrays) are not agitated, as for example may occur through an appliedexternal force that is perpendicular to the well height/depth. Moreover,once the wells are so loaded, they are not subjected to any other forcethat could dislodge the beads from the wells.

The Examples provide a brief description of an exemplary bead loadingprotocol in the context of magnetic beads. It is to be understood that asimilar approach could be used to load other bead types. The protocolhas been demonstrated to reduce the likelihood and incidence of trappedair in the wells of the flow chamber, uniformly distribute nucleic acidloaded beads in the totality of wells of the flow chamber, and avoid thepresence and/or accumulation of excess beads in the flow chamber.

In various instances, the invention contemplates that each well in theflow chamber contain only one nucleic acid loaded bead. This is becausethe presence of two beads per well will yield unusable sequencinginformation derived from two different template nucleic acids.

As part of the sequencing reaction, a dNTP will be ligated to (or“incorporated into” as used herein) the 3′ of the newly synthesizedstrand (or the 3′ end of the sequencing primer in the case of the firstincorporated dNTP) if its complementary nucleotide is present at thatsame location on the template nucleic acid. Incorporation of theintroduced dNTP (and concomitant release of PPi) therefore indicates theidentity of the corresponding nucleotide in the template nucleic acid.If no dNTP has been incorporated, no hydrogens are released and nosignal is detected at the ChemFET surface. One can therefore concludethat the complementary nucleotide was not present in the template atthat location. If the introduced dNTP has been incorporated into thenewly synthesized strand, then the chemFET will detect a signal. Thesignal intensity and/or area under the curve is a function of the numberof nucleotides incorporated (for example, as may occur in a homopolymerstretch in the template. The result is that no sequence information islost through the sequencing of a homopolymer stretch (e.g., poly A, polyT, poly C, or poly G) in the template.

Example 1

On-Chip Polymerase Extension Detected by pH Shift on an ISFET Array.

Streptavidin-coated 2.8 micron beads carrying biotinylated synthetictemplate to which sequencing primers and T4 DNA polymerase are boundwere subjected to three sequential flows of each of the fournucleotides. Each nucleotide cycle consisted of flows of dATP, dCTP,dGTP and dTTP, each interspersed with a wash flow of buffer only. Flowsfrom the first cycle are shown in blue, flows from the second cycle inred, and the third cycle in yellow. As shown in FIG. 10A, signalgenerated for both of the two dATP flows were very similar. FIG. 10Bshows that the first (blue) trace of dCTP is higher than the dCTP flowsfrom subsequent cycles, corresponding to the flow in which thepolymerase should incorporate a single nucleotide per template molecule.FIG. 10C shows that the first (blue) trace of dGTP is approximately 6counts higher (peak-to-peak) than the dGTP flows from subsequent cycles,corresponding to the flow in which the polymerase should incorporate astring of 10 nucleotides per template molecule. FIG. 10D shows that thefirst (blue) trace of dTTP is also approximately 6 counts higher(peak-to-peak) than the dTTP flows from subsequent cycles, correspondingto the flow in which the polymerase should incorporate 10 nucleotidesper template molecule.

Example 2

Sequencing in a Closed System and Data Manipulation

Sequence has been obtained from a 23-mer synthetic oligonucleotide and a25-mer PCR product oligonucleotide. The oligonucleotides were attachedto beads which were then loaded into individual wells on a chip having1.55 million sensors in a 1348×1152 array having a 5.1 micron pitch(38400 sensors per mm²). About 1 million copies of the syntheticoligonucleotide were loaded per bead, and about 300000 to 600000 copiesof the PCR product were loaded per bead. A cycle of 4 nucleotidesthrough and over the array was 2 minutes long. Nucleotides were used ata concentration of 50 micromolar each. Polymerase was the only enzymeused in the process. Data were collected at 32 frames per second.

FIG. 11A depicts the raw data measured directly from an ISFET for thesynthetic oligonucleotide. One millivolt is equivalent to 68 counts. Thedata are sampled at each sensor on the chip (1550200 sensors on a 314chip) many times per second. The Figure is color-coded for eachnucleotide flow. With each nucleotide flow, several seconds of imagingoccur. The graph depicts the concatenation of those individualmeasurements taken during each flow. The Y axis is in raw counts, andthe X axis is in seconds. Superimposed just above the X axis are theexpected incorporations at each flow.

FIG. 11 B depicts the integrated value for each nucleotide flow,normalized to the template being sequenced. The integrated value istaken from the raw trace measurements shown in FIG. 11 A, and theintegral bounds have been chosen to maximize signal to noise ratio. Theresults have been normalized to the signal per base incorporation, andgraphed per nucleotide flow. The Y axis is incorporation count, and theX axis is nucleotide flow number, alternating through TACG.

What is claimed is:
 1. A method for analyzing a sample on a sensorarray, comprising: generating a sensor signal from achemically-sensitive field effect transistor (chemFET) sensor inresponse to a reaction occurring in a reaction well capacitively coupledto the chemFET sensor; coupling a reference signal from a referencecircuit directly to a column buffer input of a column buffer connectedto a circuit node via a reference circuit switch coupled to the circuitnode in response to a timing signal during a first time interval toprovide a reference sample via the column buffer to a sampling circuit;coupling the sensor signal to the column buffer via a row select switchbetween the chemFET sensor and the circuit node; the row select switchcoupling a terminal of the chemFET sensor to the column buffer during asecond time interval; and producing an output signal of the chemFETsensor in the sampling circuit as a function of the reference sampleobtained during the first interval and a sensed sample of the sensorsignal obtained during the second interval, wherein the output signal ofthe chemFET sensor indicates a characteristic of the reaction.
 2. Themethod of claim 1, wherein the characteristic of the reaction is relatedto a concentration of a charged species in a sample fluid.
 3. The methodof claim 1, wherein the reaction occurring within the reaction well is anucleic acid sequencing reaction.
 4. The method of claim 3, wherein thecharacteristic of the reaction is related to a concentration of hydrogenions.
 5. The method of claim 1, wherein the reference signal is areference voltage.
 6. The method of claim 1, wherein producing an outputsignal further comprises: storing the output signal of the chemFETsensor in a difference circuit of the sampling circuit; and coupling thedifference circuit to a column output circuit node.
 7. The method ofclaim 1, wherein producing an output signal comprises producing anoise-reduced output signal of the chemFET sensor.